Cyclopropenones and the photochemical generation of cyclic alkynes therefrom

ABSTRACT

Cyclic alkynes (e.g., cyclooctynes such as dibenzocyclooctynes) can be photochemically generated from cyclopropenones as disclosed herein. The cyclic alkynes can be reacted (e.g., in situ) with materials having alkyne-reactive groups (e.g., azide groups in a “click” reaction). In preferred embodiments, the generation and reaction of the cyclic alkyne can proceed in the absence of a catalyst (e.g., Cu(I)). These reactions can be useful, for example, for the selective labeling of living cells that are metabolically modified with azido-containing surface monosaccharides, or for light-directed surface patterning.

This application is a divisional application of U.S. patent applicationSer. No. 12/708,617, filed Feb. 19, 2010, which claims the benefit ofU.S. Provisional Application Ser. Nos. 61/153,762, filed Feb. 19, 2009,and 61/238,835, filed Sep. 1, 2009, all of which are herein incorporatedby reference in their entireties.

GOVERNMENT RIGHTS

The present invention was made with support from the National ScienceFoundation under Grant No. CHE0449478, and the National Institutes ofHealth Grant Nos. CA88986 and GM 61761. The U.S. government has certainrights in this invention.

BACKGROUND

Connection (or ligation) of two fragments to make a larger molecule orstructure is often achieved with the help of the so-called “clickchemistry”. This term is used to describe a set of bimolecular reactionsthat meet the following criteria: reactions should be wide in scope butselective; produce high yield of the product, proceed with reasonablerate under mild conditions; and tolerate broad range of solvents. Amongknown click reactions is the reaction of azides with acetylenes. Theformation of 1,2,3-triazoles in 1,3-dipolar cycloaddition of azides totriple bonds is known, but because the activation energy ofacetylene-azide cycloaddition is relatively high (ΔG

approximately 26 kcal/mol), the reaction is very slow under ambientconditions.

The utility of the reaction of azides with alkynes was expanded by thediscovery of Cu (I) catalysis. 1,3-cycloaddition of azides to teiminalacetylenes in the presence of catalytic amounts of cuprous salts isfacile at room temperature in organic or aqueous solutions. Thecopper-catalyzed version of the acetylene- azide cycloaddition (a.k.a.azide click reaction) found a broad range of applications frommicroelectronics to virus labeling to drug development. However, the useof cytotoxic Cu (I) catalysts have largely precluded application of thisclick reaction in living systems.

Catalyst-free 1,3-dipolar cycloaddition of azides to cyclooctynes hasmade possible a bio-compatible version of the azide click reaction. Thetriple bond incorporated in an eight-membered ring is apparently alreadybent into the transition state-resembling geometry, thus reducing theactivation barrier.

Besides biocompatibility, another major bottleneck in the application ofchemical reporters in living system is the lack of spatial and temporalresolution. Photochemical immobilization of carbohydrates, proteins, DNAfragments, antibodies, and other substrates allows for the formation ofpatterned or gradient arrays on various surfaces. These techniques arewidely used in the development of novel high throughput analyticalmethods. Due to good compatibility of azide click chemistry with variousbiological substrates, and the robustness of the triazole linker, it hasbeen employed in surface functionalization including, for example,carbohydrate and protein immobilization. However, this immobilizationtechnique was not amenable to patterned modification of the surface.Although SEM-directed electrochemical reduction of Cu(II) to Cu(I)allows the patterning of fluorescent molecules on a glass slide, thismethod is of limited in scope and practicality.

New methods for ligating fragments to make a larger molecule orstructure are needed in the art.

SUMMARY

The present disclosure is generally related to methods for light-inducedligation of molecules, preferably without the use of a catalyst. Inparticular, the disclosure relates to the generation of reactiveacetylenes produced by the light-induced decarbonylation ofcyclopropenones as disclosed herein. The photochemical ligation methodof the present disclosure provides a method of linking two moleculestriggered by the photochemical generation of cyclic alkynes (e.g.,cyclooctynes) from corresponding cyclopropenones.

In one aspect, the present disclosure provides cyclopropenones andmethods of photochemically inducing the reaction of two materials usingthe cyclopropenones. In one embodiment, the method includes:photochemically generating a cyclic alkyne from a cyclopropenone; andcontacting the cyclic alkyne with a material including analkyne-reactive group (e.g., a 1,3-dipole-functional compound) underconditions effective for the cyclic alkyne and the material includingthe alkyne-reactive group to react. In some embodiments, the methodphotochemically induces the ligation of the cyclic alkyne and thematerial including the alkyne-reactive group through the formation, forexample, of a cyclic adduct (e.g., a heterocyclic compound), preferablywithout the use of a catalyst (e.g., a metal-containing catalyst).

In one embodiment, the cyclopropenone has the formula:

wherein Ar is a group representing a monocyclic or polycyclic, aromaticor heteroaromatic ring, and the dashed line represents a four atombridge. In certain embodiments, the four atom bridge includes carbonatoms, oxygen atoms, nitrogen atoms, phosphorus atoms, or combinationsthereof.

In another embodiment, the cyclopropenone has the formula:

wherein each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents NR⁶,⁺N(R⁶)₂,S, S═O, SO₂, O, PR⁶, or C(R⁴)₂; each R⁴ is independently selected fromthe group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate,nitrite, sulfate, a C1-C10 organic group, and a linking group; and eachR⁶ is independently hydrogen, a C1-C10 organic group, and/or a linkinggroup. Linking groups can be useful, for example, for attachingsubstrates and/or tags.

In another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; G represents CR⁶, N, or P;and each R⁵ and R⁶ is independently selected from the group consistingof hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, aC1-C10 organic group, and a linking group. Linking groups can be useful,for example, for attaching substrates and/or tags.

In another embodiment, the cyclopropenone have the formula:

wherein each Ar is a group independently representing a monocycle orpolycyclic, aromatic or heteroaromatic ring.

In certain embodiments, the photochemically generated cycloalkynes maythen undergo a facile “strain-promoted” cycloaddition reaction with atleast one 1,3-dipole-functional compound (e.g., an azide-functionalcompound, a nitrile oxide-functional compound, a nitrone-functionalcompound, an azoxy-functional compound, and/or an acyl diazo-functionalcompound) to form a heterocyclic compound, preferably in the absence ofadded catalyst (e.g., Cu(I)). Significantly and advantageously for theuse of the products in in vivo studies, for example, the cyclopropenoneprecursor itself does not react with 1,3-dipole-functional compound(e.g., azide functional compounds) in the absence of light, and isstable.

Thus, in another aspect, the present disclosure provides a method ofphotochemically inducing the ligation of two molecules. In oneembodiment, the method includes: (a) photochemically generating a cyclicalkyne (e.g., a cyclooctyne) from a cyclopropenone; and (b) contactingthe cyclic alkyne with an azide under conditions effective to form atriazole.

In certain embodiments, the cyclopropenone is a dibenzocyclopropenonehaving the formula I:

wherein: R¹ is selected from the group consisting of: an alkoxy and ahydroxyl; R² is selected from the group consisting of an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, an alkoxy, acarboxy, a hydroxyl, an ether, an ester, and a halogen; and thecyclooctyne is a dibenzocyclooctyne. Alternatively, or in addition to,R² can be a PEGylated group, a biotinylated group, and/or a groupcontaining an amide or carbamate linker. As used herein, the terms“PEGylated” and “biotinylated” are meant to describe groups that includea polyethylene glycol (PEG) fragment or a biotin fragment, respectively.Optionally at least one of the azide or the cyclooctyne precursor can bebound to the surface of a substrate (e.g., a solid substrate or a cellmembrane) and/or integrated into a substrate layer.

In certain embodiments, step (a) includes irradiating the cyclopropenonewith light having a wavelength (e.g., 220 nm to about 450 nm)selectively absorbed by the cyclopropenone, and substantially notabsorbed by a cyclic alkyne or by a triazole.

In certain embodiments, the method further includes the step ofproviding a cyclopropenone, said step including: (i) providing a3,3′-dialkyloxybibenzyl; and (ii) reacting the 3,3′-dialkyloxybibenzylwith tetrachloropenone in the presence of anhydrous aluminum chlorideunder medium dilution conditions effective to generate a cyclopropenone.

In another aspect, the present disclosure provides cyclopropenones. Inone embodiment, the cyclopropenones have the formula:

wherein Ar is a group representing a monocycle or polycyclic, aromaticor heteroaromatic ring, and the dashed line represents a four atombridge. In certain embodiments, the four atom bridge includes carbonatoms, oxygen atoms, nitrogen atoms, phosphorus atoms, or combinationsthereof.

In another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocycle orpolycyclic, aromatic or heteroaromatic ring; E represents NR⁶, ⁺N(R⁶)₂,S, S═O, SO₂, O, PR⁶, or C(R⁴)₂; each R⁴ is independently selected fromthe group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate,nitrite, sulfate, a C1-C10 organic group, and a linking group; and eachR⁶ is independently hydrogen, a C1-C10 organic group, and/or a linkinggroup. Linking groups can be useful, for example, for attachingsubstrates and/or tags.

In another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocycle orpolycyclic, aromatic or heteroaromatic ring; G represents CR⁶, N, or P;and each R⁵ and R⁶ is independently selected from the group consistingof hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, aC1-C10 organic group, and a linking group. Linking groups can be useful,for example, for attaching substrates and/or tags.

In still another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring. In certain preferredembodiments, the cyclopropenone has the formula I:

wherein: R¹ is selected from the group consisting of: an alkoxy and ahydroxyl; R² is selected from the group consisting of: an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, an alkoxy, acarboxy, a hydroxyl, an ether, an ester, and a halogen; and thecyclooctyne is a dibenzocyclooctyne. Alternatively, or in addition to,R² can be a PEGylated group, a biotinylated group, and/or a groupcontaining an amide or carbamate linker.

In another aspect, the present disclosure provides a dibenzocyclooctyneof the formula:

wherein: R¹ is selected from the group consisting of: an alkoxy and ahydroxyl; R² is selected from the group consisting of: an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, an alkoxy, acarboxy, a hydroxyl, an ether, an ester, and a halogen; and thecyclooctyne is a dibenzocyclooctyne. Alternatively, or in addition to,R² can be a PEGylated group, a biotinylated group, and/or a groupcontaining an amide or carbamate linker.

In yet another aspect, the present disclosure provides a triazole of thefoiniula:

wherein: R¹ is selected from the group consisting of: an alkoxy and ahydroxyl; R² is selected from the group consisting of: an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, an alkoxy, acarboxy, a hydroxyl, an ether, an ester, and a halogen; and R³ isselected from the group consisting of a primary alkyl, a secondaryalkyl, a tertiary alky, an aryl, an alkylaryl, an acyl, an alkylacyl,and an arylacyl. Alternatively, or in addition to, R² can be a PEGylatedgroup, a biotinylated group, and/or a group containing an amide orcarbamate linker.

In other certain embodiments, the photochemically generated cycloalkynesmay then undergo cycloaddition reactions (e.g., thermally promotedreactions) with dienes to give Diels-Alder adducts; with nitrosoarenesto give N-hydroxy indoles; with an alkene and a metal carbene complex togive butadiene products (e.g., enyne metathesis); with alkynes and ametal catalyst to give new alkynes (e.g., alkyne metathesis); with othermetal-containing compounds such as, for example, four- and/orfive-membered platinacycles to give cycloaddition products; with alkenesand carbon monoxide to give [2+2+1] cycloaddition products (e.g., aPauson Khand reaction); with compounds bearing intermetallic multiplebonds (e.g., (η-C₅Me₅)₂Rh₂(μ-CO)₂, [RO]₃Mo≡Mo[OR]₃, [RCO₂]₂W

W[O₂CR]₂, complexes with a double, triple and quadruple metal-metalbond, respectively) to yield, for instance, terminal (M≡CR) or bridged(M-C(R)-M] metal-carbido, or bridging alkyne complexes; and withnitriles, cyanates, isocyanates, and/or isothiocyanates, under theappropriate conditions, to yield the respective metathesis and/orcycloaddition products.

The term “cycloaddition” as used herein refers to a chemical reaction inwhich two or more pi-electron systems (e.g., unsaturated molecules orunsaturated parts of the same molecule) combine to form a cyclic productin which there is a net reduction of the bond multiplicity. In acycloaddition, the pi-electrons are used to form new sigma bonds. Theproduct of a cycloaddition is called an “adduct” or “cycloadduct”.Different types of cycloadditions are known in the art including, butnot limited to, [3+2] cycloadditions and Diels-Alder reactions. [3+2]cycloadditions, which are also called 2,3-dipolar cycloadditions, occurbetween a 1,3-dipole and a dipolarophile and are typically used for theconstruction of five-membered heterocyclic rings.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “comprises,”“comprising,” “containing” and “having” and the like can have themeaning ascribed to them in U.S. Patent law and can mean “includes,”“including,” and the like; “consisting essentially of” or “consistsessentially” or the like, when applied to methods and compositionsencompassed by the present disclosure refers to compositions like thosedisclosed herein, but which may contain additional structural groups,composition components or method steps (or analogs or derivativesthereof as discussed above). Such additional structural groups,composition components or method steps, etc., however, do not materiallyaffect the basic and novel characteristic(s) of the compositions ormethods, compared to those of the corresponding compositions or methodsdisclosed herein. “Consisting essentially of” or “consists essentially”or the like, when applied to methods and compositions encompassed by thepresent disclosure have the meaning ascribed in U.S. Patent law and theterm is open-ended, allowing for the presence of more than that which isrecited so long as basic or novel characteristics of that which isrecited is not changed by the presence of more than that which isrecited, but excludes prior art embodiments.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. The term “about” can include ±1%, ±2%, ±3%, ±4%, ±5%,±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) beingmodified.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying figures.

FIG. 1 illustrates a generalized scheme for the photochemical generationof a dibenzocyclooctyne from a cyclopropenone precursor followed byreaction with an azide to produce a triazole.

FIG. 2 illustrates an exemplary embodiment in which cyclopropenone-based“photo-click” chemistry can be used to label living organisms.

FIG. 3 a illustrates a generalized scheme for photochemical initiationof a acetylene-azide cycloaddition, preferably without the use of acopper catalyst. FIG. 3 b illustrates the structure of knowndibenzocyclooctynes.

FIG. 4 illustrates a generalized scheme for the preparation of variouscyclopropenones. Reagents and conditions: a) AlCl₃; b)HO(CH₂)₂O(CH₂)₂OAc, PPh₃, DEAD, THF; c) neopentyl glycol, BF₄O(C₂H₅)₃,Et₃N, CH₂Cl₂; d) NaOH, MeOH; e) p-nitrophenyl chloroformate, pyridine;f) N-Boc-N′-biotinyl-3,6-dioxaoctane-1,8-diamine, Et₃N, DMF; g)Amberyst-15 H⁺, acetone.

FIG. 5 illustrates an independent preparation of biotinylated acetylene6b. Reagents and conditions. a) 350 nm irradiation, MeOH-THF; b) NaOH,MeOH; c) p-nitrophenyl chloroformate, pyridine; d)N-Boc-N′-biotinyl-3,6-dioxaoctane-1,8-diamine, Et₃N, DMF.

FIG. 6 is a graph illustrating the spectra of (about 5×10⁻⁵ M) methanolsolutions of cyclopropenone 5c ; acetylene 6c ; and triazole 7c.

FIG. 7 illustrates cell surface labeling with compounds 4b, 5b, and 6b.Jurkat cells grown for 3 days in the presence of a) Ac₄ManNAc (25micromolar) or a-c) Ac₄ManNAz (25 micromolar) were incubated at roomtemperature with compounds 4b, 5b, and 6b at a) 30 micromolar for 1hour, b) 0-100 micromolar for 1 hour, or c) 30 micromolar for 0-90minutes. Compound 5b was assessed without activation (5b NA) and afterlight activation in situ (1 minute at 350 nm; 5b IS). Next, cells wereincubated with avidin-FITC for 15 minutes at 4° C., after which celllysates were assessed for fluorescence intensity. AU indicates arbitraryfluorescence units.

FIG. 8 illustrates a Western blot analysis of cell surface labeling withcompounds 5b and 6b. Jurkat cells grown for 3 days in the presence ofAc₄ManNAc (25 micromolar; lanes 1, 3, and 5) or Ac₄ManNAz (25micromolar; lanes 2, 4, and 6) were incubated with 5b (lanes 3 and 4),6b (lanes 5 and 6) (30 micromolar), or without compound (lanes 1 and 2)for 1 hour at room temperature. Compound 5b was assessed after lightactivation in situ (5b IS). Cell lysates (20 micrograms total proteinper lane) were resolved by SDS-PAGE and the blot was probed with ananti-biotin antibody conjugated to HRP. Total protein loading wasconfirmed by Coomassie staining of the gel (not shown).

FIG. 9 is a graph illustrating a toxicity assessment of cycloadditionreaction with compound 5b. Jurkat cells grown for 3 days in the presenceof Ac₄ManNAz (25 micromolar) were incubated with compound 5b (30micromolar) for 1 hour at room temperature. Cell viability afterincubation with 5b was assessed without activation (5b NA; III) andafter light activation in situ (1 minute at 350 nm; 5b IS; IV). Controlcells were treated similarly, but without exposure to 5b and UV light(II). Cell viability was assessed with trypan blue exclusion. Cellviability values were normalized for the amount of viable cells of thesample with control cells before the incubation period (100%; I).

FIG. 10 illustrates exemplary fluorescence images of cells labeled withcompound 5b and avidin-Alexa fluor 488. CHO cells grown for 3 days inthe presence of Ac₄ManNAc (100 micromolar; A) or Ac₄ManNAz (100micromolar; B) were given compound 5b (30 micromolar), subjected to 1minute of UV light for in situ activation (5b IS), and further incubatedfor 1 hour at room temperature. Next, cells were incubated withavidin-Alexa Fluor 488 for 15 minutes at 4° C. and, after washing,fixing, and staining for the nucleus with the far-red-fluorescent dyeTO-PRO-3 iodide, imaged. Merged indicate that the images of cellslabeled with Alexa Fluor (488 nm) and TO-PRO iodide (633 nm) are mergedand shown in white and gray, respectively.

FIG. 11 illustrates a scheme for the synthesis of embodiments of atriazole from a dibenzocyclooctyne, where the R substituent on thetriazole is defined.

FIG. 12 illustrates a generalized scheme for cyclopropenone synthesis,where substituents may be attached to the cyclopropenone precursor byreplacement of a hydroxyl group with a linker The reaction of 5a withbutanol or diethylene glycol acetate in the presence of PPh₃ and DEAD at0° C. produces 5c and 5d. The reaction of 5a with a carboxylic acid inthe presence of DCC and catalytic amount of DMAP provided ester 5e.Diethylene glycol-derivatized cyclopropenone 5d can be further linked tobiotin, producing the biotin-cyclopropenone conjugate 5b.

FIG. 13 illustrates a generalized scheme for the preparation ofcyclopropenones 5c and 5a. Reagents and conditions for the reactionswere a) BBr₃, CH₂Cl₂; then BuBr, K₂CO₃, DMF; 72% over 2 steps; and b)AlCl₃, CH₂Cl₂, 35%.

FIGS. 14-16 illustrate exemplary methods for preparing cyclopropenonesas further described herein.

The drawings are described in greater detail in the description andexamples below.

The details of some exemplary embodiments of the methods and systems ofthe present disclosure are set forth in the description below. Otherfeatures, objects, and advantages of the disclosure will be apparent toone of skill in the art upon examination of the following description,drawings, examples and claims. It is intended that all such additionalsystems, methods, features, and advantages be included within thisdescription, be within the scope of the present disclosure, and beprotected by the accompanying claims.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The methods of the present disclosure provide for photochemicallyinducing the reaction of two materials by photochemically generating anactivated alkyne (e.g., a cyclooctyne) from a cyclopropenone. Thegenerated cyclic alkyne can react with another material, which incertain embodiments causes ligation of the cyclic alkyne with the othermaterial.

The term “activated alkyne,” as used herein, refers to a chemical groupthat selectively reacts with an alkyne-reactive group, such as an azidogroup or a phosphine group, on another molecule to form a covalentchemical bond between the activated alkyne group and the alkyne reactivegroup. Examples of alkyne-reactive groups include azides.“Alkyne-reactive” can also refer to a molecule that contains a chemicalgroup that selectively reacts with an alkyne group. As used herein“activated alkyne” encompasses any terminal alkynes or cyclic alkynes(dipolarophiles) that will react with 1,3-dipoles such as azides in afacile fashion.

The term “azide reactive,” as used herein, refers to a material thatselectively reacts with an azido modified group on another molecule toform a covalent chemical bond between the azido modified group and theazide reactive group. Examples of azide-reactive groups include alkynesand phosphines (e.g., triaryl phosphine). “Azide-reactive” can alsorefer to a molecule that selectively reacts with an azido group.

In certain embodiments, the photochemically generated cycloalkynes maythen undergo a facile “strain-promoted” cycloaddition reaction with atleast one 1,3-dipole-functional compound (e.g., an azide-functionalcompound, a nitrile oxide-functional compound, a nitrone-functionalcompound, an azoxy-functional compound, and/or an acyl diazo-functionalcompound) to foam a heterocyclic compound, preferably in the absence ofadded catalyst (e.g., Cu(I)). Significantly and advantageously for theuse of the products in in vivo studies, for example, the cyclopropenoneprecursor itself does not react with 1,3-dipole-functional compounds(e.g., azide functional compounds) in the absence of light, and arestable, capable of withstanding prolonged heating.

In other certain embodiments, the photochemically generated cycloalkynesmay then undergo cycloaddition reactions (e.g., thermally promotedreactions) with dienes to give Diels-Alder adducts; with nitrosoarenesto give N-hydroxy indoles; with an alkene and a metal carbene complex togive butadiene products (e.g., enyne metathesis); with alkynes and ametal catalyst to give new alkynes (e.g., alkyne metathesis); with othermetal-containing compounds such as, for example, four-and/orfive-membered platinacycles to give cycloaddition products; and withalkenes and carbon monoxide to give [2+2+1] cycloaddition products(e.g., a Pauson Khand reaction).

The methods encompassed by the present disclosure may be useful forgenerating surfaces, modified with cyclopropenone-containing compounds,which may be used for the patterned immobilization of a broad range ofbiomolecules.

The present disclosure provides a method of photochemically inducing theligation of two molecules, the method including: (a) photochemicallygenerating a cyclic alkyne (e.g., a cyclooctyne) from a cyclopropenone;and (b) contacting the cyclic alkyne with an azide under conditionseffective to form a triazole.

In some embodiments, the cyclopropenones have the formula:

wherein Ar is a group representing a monocyclic or polycyclic, aromaticor heteroaromatic ring, and the dashed line represents a four atombridge. In certain embodiments, the four atom bridge includes carbonatoms, oxygen atoms, nitrogen. atoms, phosphorus atoms, or combinationsthereof. Such cyclopropenones can be prepared, for example, by theaddition of a dihalocarbene to a corresponding cyclic alkyne followed byhydrolysis in methods similar to those further described herein below.

In another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocycle orpolycyclic, aromatic or heteroaromatic ring; E represents NR⁶, ⁺N(R⁶)₂,S, S═O, SO₂, O, PR⁶, or C(R⁴)₂; each R⁴ is independently selected fromthe group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate,nitrite, sulfate, a C1-C10 organic group, and a linking group; and eachR⁶ is independently hydrogen, a C1-C10 organic group, and/or a linkinggroup. Linking groups can be useful, for example, for attachingsubstrates and/or tags. In some embodiments, such cyclopropenones can beprepared, for example, via a double Friedel-Crafts alkylation asillustrated, for example, in FIG. 14. In other embodiments, suchcyclopropenones can be prepared, for example, by the addition of adihalocarbene to a corresponding cyclic alkyne followed by hydrolysis asillustrated, for example, in FIG. 15. See, for example, Poloukhtine etal., Chemical Communications 2005, 617-619; and Kuzmin et at, ChemicalCommunications 2009, 5707-5709. See, also, Poloukhtine et al., Journalof Organic Chemistry 2005, 70(4):1297-1305.

In another embodiment, the cyclopropenones have the formula:

wherein each Ar is a group independently representing a monocycle orpolycyclic, aromatic or heteroaromatic ring; G represents CR⁶, N, or P;and each R⁵ and R⁶ is independently selected from the group consistingof hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, aC1-C10 organic group, and a linking group. Linking groups can be useful,for example, for attaching substrates and/or tags. In some embodiments,such cyclopropenones can be prepared, for example, via a doubleFriedel-Crafts alkylation as illustrated, for example, in FIG. 16.

In another embodiment, the cyclopropenone has the formula:

wherein each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring.

As used herein, the term “organic group” is used for the purpose of thisdisclosure to mean a hydrocarbon group that is classified as analiphatic group, cyclic group, or combination of aliphatic and cyclicgroups (e.g., alkaryl and aralkyl groups). In the context of the presentdisclosure, suitable organic groups for cyclopropenones and compoundshaving alkyne reactive groups as disclosed herein are those that do notinterfere with the photochemical generation of the cyclic alkyne or thereaction of the cyclic alkyne with a compound having an alkyne reactivegroup. In the context of the present disclosure, the term “aliphaticgroup” means a saturated or unsaturated linear or branched hydrocarbongroup. This term is used to encompass alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a saturated linear orbranched monovalent hydrocarbon group including, for example, methyl,ethyl, n-propyl, isopropyl, tert-butyl, amyl, heptyl, and the like. Theterm “alkenyl group” means an unsaturated, linear or branched monovalenthydrocarbon group with one or more olefinically unsaturated groups(i.e., carbon-carbon double bonds), such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched monovalenthydrocarbon group with one or more carbon-carbon triple bonds. The term“cyclic group” means a closed ring hydrocarbon group that is classifiedas an alicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polynuclear aromatic hydrocarbon group. The term“heterocyclic group” means a closed ring hydrocarbon in which one ormore of the atoms in the ring is an element other than carbon (e.g.,nitrogen, oxygen, sulfur, etc.).

As a means of simplifying the discussion and the recitation of certainterminology used throughout this application, the terms “group” and“moiety” are used to differentiate between chemical species that allowfor substitution or that may be substituted and those that do not soallow for substitution or may not be so substituted. Thus, when the term“group” is used to describe a chemical substituent, the describedchemical material includes the unsubstituted group and that group withnonperoxidic O, N, S, Si, or F atoms, for example, in the chain as wellas carbonyl groups or other conventional substituents. Where the term“moiety” is used to describe a chemical compound or substituent, only anunsubstituted chemical material is intended to be included. For example,the phrase “alkyl group” is intended to include not only pure open chainsaturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like, but also alkyl substituents bearing furthersubstituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group”includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,hydroxyalkyls, snlfoalkyls, etc. On the other hand, the phrase “alkylmoiety” is limited to the inclusion of only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like.

The term azide as used herein refers to organic azides having thegeneral formula R—N₃ where R is an organic group selected from the groupconsisting of alkyl, alkyl amino, nitrogen-containingheterocyclic-substituted alkyl (that is, an alkyl group substituted withat least one nitrogen-containing heterocycle), and alkyl aminesubstituted with at least one alkyl azide group. Non-limiting examplesof alkyl groups include methyl, ethyl, propyl, butyl, and isomers (iso-,sec-, tert-, etc.) thereof. Non-limiting examples of alkyl amino groupsinclude dimethylamino, diethylamino, dipropylamino, dibutylamino, andisomers thereof, as well as “mixed” alkyl amino groups, e.g., N-methyl,N-ethylamino; N-propyl, N-butylamino; etc.; and isomers thereof.Non-limiting examples of nitrogen-containing heterocyclic-substitutedalkyl groups include alkyl groups substituted with pyrrollidine,imidazole, pyrrole, piperidine, pyrroline, pyrazole, piperazine, or1,2,4-triazole. When R is an “alkyl amine substituted with at least onealkyl azide group” the organic azide has the formula R¹NH(R²N₃) orR¹N(R²N₃)(R³N₃), where R¹, R², and R³ are each, independently, an alkylgroup as described above. A non-limiting example of such a compound isbis(ethylazide) methylamine. The organic azides referred to herein have,in each case, a carbon atom bound directly to one of the nitrogen atomsof the azide (N₃) group. Hence, in some cases, it may be moreappropriate to refer to the alkyl groups as “alkylenyl” groups.

As illustrated in FIG. 1, for example, dibenzocyclooctynes 2 can begenerated by the photo-induced decarbonylation reaction of thecorresponding cyclopropenones 1. The dibenzocyclooctynes 2 can thenundergo facile reactions with azides preferably to produce quantitativeyields of a corresponding triazole 3. It is contemplated that a widevariety of substituents (R¹, R² , etc) can be introduced into thearomatic rings of 1 to serve as linkers to substrates of interest, orremain as a substrate of interest themselves. It is contemplated,however, that a preferred group R¹ can be either a hydroxyl or an alkoxygroup, and in certain embodiments, any substituent linked to an aromaticring of the dibenzocyclooctyne or dibenzocyclooctyne precursor is not astrong electron withdrawing group such as, but not limited to, a nitrogroup, a carbonyl group, and a cyano group. It is further contemplatedthat for certain embodiments, a bulky group (R²) is not linked to aposition on an aromatic ring of the dibenzocyclooctyne ordibenzocyclooctyne precursor that is ortho to an alkyne orcyclopropenone substituent of the aromatic ring.

Thus, the method of the present disclosure is a two-step procedure wherethe “click chemistry” that allows the conjugation of the cyclic alkyne(e.g., a cyclooctyne) with an azide is preceded by the light-inducibleformation of the cyclic alkyne, which provides a selective means ofinitiating the overall pathway, under conditions conducive to their usein living cells without toxic effects from such as cuprous catalysts.The term “click chemistry,” as used herein, refers to the Huisgencycloaddition or the 2,3-dipolar cycloaddition between an azide and aterminal alkyne to form a 1,2,4-triazole. Such chemical reactions canuse, but are not limited to, simple heteroatomic organic reactants andare reliable, selective, stereospecific, and exothermic. In theembodiments of the methods encompassed by the present disclosure, theconversion of a cyclopropenone to a cyclic alkyne can be induced by alight source such as, but not limited to, a laser light having awavelength of from about 220 nm to about 450 nm or even longer (e.g.,350 nm, 405 nm, and 425 nm), from about 325 nm to about 375 nm, fromabout 325 nm to about 355 nm, and from about 350 nm to about 355 nm. Useof light (laser or non-laser) with a wavelength from about 340 nm toabout 375 nm, for example, is desirable when such as dibenzocyclooctyneor the triazole do not absorb light at these wavelengths. Thelight-inducible reaction, therefore, provides for initiating ortriggering the reaction when desired, and focusing of the laser lightmay allow triggering of the reaction, and therefore the coupling betweenthe cyclopropenone and the azide, at a specific and confined location,such as, for example, a single cell, or at a previously selectedlocation within a cell.

The present disclosure, therefore, provides embodiments of a method ofphotochemically inducing the ligation of two molecules, the methodincluding: (a) photochemically generating a cyclic alkyne (e.g.,cyclooctyne) from a cyclopropenone; and (b) contacting the cyclic alkynewith an azide under conditions effective to form a triazole.

In the embodiments of the methods of the present disclosure, thecyclopropenone may be a dibenzocyclopropenone having the formula I:

where R¹ can be selected from the group consisting of: an alkoxy and ahydroxyl, and R² can be a substituent, and where, when the cyclooctyneis a dibenzocyclooctyne, R² is selected from the group consisting of: analkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, analkoxy, a carboxy, a hydroxyl, an ether, an ester, and a halogen.Alternatively, or in addition to, R² can be a PEGylated group, abiotinylated group, and/or a group containing an amide or carbamatelinker. The cyclooctyne can be a dibenzocyclooctyne.

In these embodiments of the disclosure, R¹ and R² can each beindependently linked to any available position of an aromatic ring ofthe dibenzocyclooctyne or dibenzocyclooctyne precursor.

The compounds described herein may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL's ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5.sup.TH ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Ace. Chem. Res. 23: 128 (1990).

Where a disclosed compound includes a conjugated ring system, resonancestabilization may permit a formal electronic charge to be distributedover the entire molecule. While a particular charge may be depicted aslocalized on a particular ring system, or a particular heteroatom, it iscommonly understood that a comparable resonance structure can be drawnin which the charge may be formally localized on an alternative portionof the compound.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH therefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present disclosure contain betweenabout one and about twenty five carbon atoms (e.g., methyl, ethyl andthe like). Straight, branched or cyclic hydrocarbon chains having eightor fewer carbon atoms will also be referred to herein as “lower alkyl”.In addition, the term “alkyl” as used herein further includes one ormore substitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The term “amino” or “amine group” refers to the group —NR′R″ (orN⁺RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, including thegroup N⁺RR′R″ and its biologically compatible anionic counterions.

The term “aryl” as used herein refers to a cyclic aromatic carbon chainhaving twenty or fewer carbon atoms, e.g., phenyl, naphthyl, biphenyl,and anthracenyl. One or more carbon atoms of the aryl group may also besubstituted with, e.g., alkyl; aryl; heteroaryl; a halogen; nitro;cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl-, orarylthio; amino, alkylamino, arylamino, dialkyl-, diaryl-, orarylalkylamino; aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl In addition, two ormore alkyl or heteroalkyl substituents of an aryl group may be combinedto form fused aryl-alkyl or aryl-heteroalkyl ring systems (e.g.,tetrahydronaphthyl). Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The terms “alkoxy,” “alkylamino”, and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P, S, and Se, and wherein thenitrogen, phosphorous, sulfur, and selenium atoms are optionallyoxidized, and the nitrogen heteroatom is optionally be quaternized. Theheteroatom(s) O, N, P, S, Si, and Se may be placed at any interiorposition of the heteroalkyl group or at the position at which the alkylgroup is attached to the remainder of the molecule. Examples include,but are not limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited, by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic group that can be a single ring or multiple rings (preferablyfrom 1 to 3 rings), which are fused together or linked covalently. Theterm “heteroaryl” refers to aryl groups (or rings) that contain from oneto four heteroatoms selected from N, O, S, and Se, wherein the nitrogen,sulfur, and selenium atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxo1-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″′″, —NR″C(O)₂R,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR″, —S(O)R′, —S(O) ₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compoundincludes more than one R group, for example, each of the R groups isindependently selected as are each R′,

R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(N⁺R′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are preferably independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR')q-U-, wherein T and U are independently —NR—, —O—, —CRR′—or a single bond, and q is an integer of from 0 to 3. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH₂)r-B-,wherein A and B are independently —CRR+—, —O—, —NR—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to4. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)s-X-(CR″R″)d-, where s and dare independently integers of from 0 to 3, and X is —O—, —NR′—, —S—,—S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R″ arepreferably independently selected from hydrogen or substituted orunsubstituted (C1-C6)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P), silicon (Si), and selenium (Se).

The term “amino” or “amine group” refers to the group —NR′R″ (or N³⁰RR′R″) where R, R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, aryl alkyl, substituted aryl alkyl, heteroaryl, and substitutedheteroaryl. A substituted amine being an amine group wherein R′ or R″ isother than hydrogen. In a primary amino group, both R′ and R″ arehydrogen, whereas in a secondary amino group, either, but not both, R′or R″ is hydrogen. In addition, the terms “amine” and “amino” caninclude protonated and quaternized versions of nitrogen, including thegroup —N³⁰ RR′R″ and its biologically compatible anionic counterions.

The term “carboxyalkyl” as used herein refers to a group having thegeneral formula —(CH₂)_(n)COOH, where n is 1-18.

The term “linking group” is broadly used herein to refer to any organic(e.g., hydrocarbon) or inorganic (e.g., N, P, O) group that can be usedfor attaching another group (e.g., a substrate or tag).

In embodiments of the disclosure, the azide or the cyclic alkyne (e.g.,a cyclooctyne) may be bound to the surface of a substrate. In theseembodiments, the substrate may be a solid substrate or a cell membrane.In other embodiments, the azide or the cyclic alkyne may be integratedinto a substrate layer.

In these embodiments, if the azide is bound to the surface of asubstrate or integrated into a substrate layer, then the cyclic alkyne(e.g., a cyclooctyne) is a ligand that binds to the azide; and wherein,if the cyclic alkyne is bound to the surface of a substrate orintegrated into a substrate layer, then the azide is a ligand that bindsto the cyclic alkyne.

In another embodiment of the disclosure, the azide ligand or the cyclicalkyne ligand (e.g., a cyclooctyne) is a detectable label.

In some embodiments of the method of the disclosure, in thecyclopropenone of formula I, R¹ may be a butoxy group and R² may beselected from the group consisting of the formulae:

In some other embodiments of the method of the disclosure, in thecyclopropenone of formula I, R¹ may be a butoxy group and R² may be aPEGylated or biotinylated group. In certain embodiments, thebiotinylated group has the formula:

In other embodiments of the method of the disclosure, the cyclopropenonemay have the formula II:

In the embodiments of the method of the disclosure, the azide may beselected from the group consisting of an alkyl azide, a heteroalkylazide, a cycloalkyl azide, a heterocycloalkyl azide, an alkylaminoazide, a benzyl azide, an aryl azide an alkylacyl azide, and an arylacylazide.

In embodiments of the method of the present disclosure, step (a)includes irradiating the cyclopropenone with light having a wavelengthselectively absorbed by the cyclopropenone, and substantially notabsorbed by a cyclic alkyne (e.g., a cyclooctyne) or by a trizaole. Theterm “substantially not absorbed” as used herein refers to the degree towhich a wavelength, or range of wavelengths, of light is absorbed by onecompound when compared with another compound. In particular, the term“substantially not absorbed” as used in the embodiments of the presentdisclosure, therefore, indicates that a cyclic alkyne or triazole willabsorb less than about 20%, advantageously less than about 10%, moreadvantageously less than about 5%, and most advantageously about 0% ofthe light absorbed by a cyclopropenone that has a wavelength able toinitiate the conversion of the cyclopropenone to a cyclic alkyne.

In the embodiments of the methods encompassed by the present disclosure,the wavelength of light is from about 220 nm to about 450 nm or evenlonger (e.g., 350 nm, 405 nm, and 425 nm). In certain embodiments of thedisclosure, the wavelength of light may be from about 325 nm to about375 nm. In other embodiments, the wavelength of light may be from about325 nm to about 360 nm. In yet other embodiments, the wavelength oflight may be from about 350 nm to about 355 nm. In still otherembodiments, the wavelength of light may be from about 340 nm to about355 nm.

In embodiments of the method of the present disclosure, the method mayfurther include the step of providing a cyclooctyne, said stepincluding: (i) providing a 3,3′-dialkyloxybibenzyl; and (ii) reactingthe 3,3′-dialkyloxybibenzyl with tetrachloropenone in the presence ofanhydrous aluminum chloride under medium dilution conditions effectiveto form a cyclopropenone.

In one embodiment of the method of the disclosure, the3,3′-dialkyloxybibenzyl is 3,3′-dibutoxybibenzyl, and the cyclopropenonehas the formula I:

where R¹ is selected from the group consisting of: an alkoxy and ahydroxyl, and R² is a substituent. In these embodiments of the methodsof the disclosure, R² may be selected from the group consisting of: analkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, analkoxy, a carboxy, a hydroxyl, an ether, an ester, and a halogen.Alternatively, or in addition to, R² can be a PEGylated group, abiotinylated group, and/or a group containing an amide or carbamatelinker.

In some embodiments of the method of the disclosure, the yield of thereaction in step (ii) may include a compound having the formula II and acompound having the formula III:

In certain embodiments of the method of the disclosure, the azide mayhave the formula:R³—N₃,where R³ may be selected from the group consisting of an an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an alkylamino, an aryl,an alkylacyl, and an arylacyl.

In certain embodiments of the method of the present disclosure, thetriazole may have the formula:

where R¹ can be selected from the group consisting of: an alkoxy and ahydroxyl; R² can be selected from the group consisting of: an alkyl, aheteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, an alkoxy, acarboxy, a hydroxyl, an ether, an ester, and a halogen; and wherein R³can be selected from the group consisting of a primary alkyl, asecondary alkyl, a tertiary alky, an aryl, an alkylaryl, an acyl, analkylacyl, and an arylacyl.

In some embodiments, R² may be selected from the group consisting of: analkyl, a heteroalkyl, a cycloalkyl, a heterocycloalkyl, an aryl, analkoxy, a carboxy, a hydroxyl, an ether, an ester, and a halogen.Alternatively, or in addition to, R² can be a PEGylated group, abiotinylated group, and/or a group containing an amide or carbamatelinker.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentdisclosure to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

Biomaterials

Photo-triggering of the azide to acetylene cycloaddition reaction (e.g.,preferably metal-free) was achieved by masking the triple bond ofdibenzocyclooctynes as cyclopropenone. Such masked cyclooctynes do notreact with azides in the dark. Irradiation of cyclopropenones results inthe efficient (Φ₃₅₅=0.33) and clean regeneration of the correspondingdibenzocyclooctynes, which then undergo facile cycloadditions (e.g.,catalyst-free cycloadditions) with azides to give correspondingtriazoles under ambient conditions. In-situ light activation of acyclopropenone linked to biotin made it possible to label living cellsexpressing glycoproteins containing N-azidoacetyl-sialic acid. Asillustrated in FIG. 2, the cyclopropenone-based “photo-click” chemistryoffers exciting opportunities to label living organisms in a temporaland for spatial controlled and may facilitate the preparation ofmicroarrays.

The bioorthogonal chemical reporter strategy is emerging as a versatilemethod for labeling of biomolecules such as nucleic acids, lipids,proteins, and carbohydrates. In this approach, a unique chemicalfunctionality is incorporated into a targeted biomolecule, preferably bythe biosynthetic machinery of the cell, followed by a specific chemicalreaction of the functional group with an appropriate probe. Inparticular, the azide is an attractive chemical reporter because of itssmall size, diverse mode of reactivity, and bio-orthogonality. Azidescan be incorporated into biomolecules using a variety of strategies suchas post synthetic modification, in-vitro enzymatic transfer, the use ofcovalent inhibitors, and metabolic labeling by feeding cells abiosynthetic precursor Modified with an azido function.

The most commonly employed bioorthogonal reactions with azides includethe Staudinger ligation with phosphines, copper(I)-catalyzedcycloaddition with terminal alkynes, and strain-promoted cycloadditionwith cyclooctynes. The latter type of reaction, which was coinedcopper-free click chemistry, does not require a cytotoxic metalcatalyst, which can therefore offer a unique opportunity for labelingliving cells. The attraction of this type of technology was elegantlydemonstrated by a study of the Bertozzi laboratory in which glycans ofthe developing zebrafish were imaged using a difluorinated cyclooctynederivative (e.g., Laughlin et al., Science 2008, 320:664-667). Boons andcoworkers have demonstrated that derivatives of 4-dibenzocyclooctynol(4a,b; DIBO, FIG. 3) react exceptionally fast in the absence of a Cu^(I)catalyst with azido-containing saccharides and amino acids, and can beemployed for visualizing glycoconjugates of living cells that aremetabolically labeled with azido-containing monosaccharides (e.g., Ninget al., Angew. Chem. Int. Ed. 2008, 47:2253-2255).

The utility of azide-based bioorthogonal reporter strategy can befurther extended by the development of a photochemically-triggered clickreaction, as this approach allows for the spatial and temporal controlof the labeling of the target substrates. In fact, photochemical releaseor generation of an active molecule is a widely employed strategy todeliver bioactive compounds to small, addressable target sites in atime-controlled manner (e.g., Pelliccioli et al., Photochem. Photobiol.Sci. 2002, 1:441-458; Mayer et al., Angew. Chem. Int. Ed. 2006,45:4900-4921; Ellis-Davies, Nat. Methods 2007, 4:619-628; and Song etal., J. Am. Chem. Soc. 2008, 130:9654-9655). To achieve this goal, wehave explored photochemical generation of reactive dibenzocyclooctynes.It is known that single (e.g., Kuzmanich et al., J. Am. Chem. Soc. 2008,130:1140-1141; Chiang et al., J. Phys. Org. Chem. 1996, 9:361-370;Dehmlow et al., Chem. Ber. 1988, 121:569; Murata et al., J. Am. Chem.Soc. 1993, 115:4013-4023; Chapman et al., J. Am. Chem. Soc. 1981, 103,7033-7036; and Poloukhtine et al., J. Org. Chem. 2003, 68:7833-7840) ortwo-photon (e.g., Urdabayev et al., Chem. Commun. 2006, 454-456)excitation of cyclopropenones results in the formation of correspondingacetylenes. Photochemical decarbonylation of thermally stablediaryl-substituted cyclopropenones is especially efficient (Φ=0.6−1.0)and produces alkynes in a quantitative yield (e.g., Poloukhtine et al.,J. Org. Chem. 2003, 68:7833-7840). This reaction is also extremely fastand is complete within few hundred picoseconds after excitation (e.g.,Poloukhtine et al., J Phys. Chem. A 2006, 110:1749-1757). We havealready employed cyclopropenone groups in the development ofphotoswitchable enediynes (e.g., Poloukhtine et al., Chem. Commun. 2005,617-619; Poloukhtine et al., J. Org. Chem. 2005, 70:1297-1305;Poloukhtine et al., J. Org. Chem. 2006, 71:7417-7421; and Pandithavidanaet al., J. Am. Chem. Soc. 2009, 131:351-356). Here we report a novel“photo-click” strategy for the ligation of azides, which in preferredembodiments is metal-free (FIG. 3). Cyclopropenones, such as 5 , do notreact with azides under ambient conditions in the dark but efficientlyproduce reactive dibenzocyclooctynes 6 upon irradiation. The latter typeof compound could be employed for labeling of living cells modified withazido-containing cell surface saccharides.

Interestingly, the rate constants for cycloaddition of acetylene 6a-cwith benzyl- and phenyl azide at 25±0.1° C. were very similar to that ofdibenzocyclooctynol (4a) (e.g., Ning et al., Angew. Chem. Int. Ed. 2008,47:2253-2255), and thus, the aromatic alkoxy-substitutents of 6a-c donot appear to influence the rate constants (6c: PhN₃ 0.0163±0.0006M⁻¹s⁻¹; BnN₃ 0.0763±0.0011 M⁻¹s⁻¹; 4a: 0.0567±0.0027 M⁻¹s⁻¹ and 0.17M⁻¹s⁻¹) (e.g., Ning et al., Angew. Chem. Int. Ed. 2008, 47:2253-2255).

Synthesis of Cyclopropenones 5a-c and Acetylene 6b.

Friedel-Crafts alkylation of appropriate substrates withtricholorocyclopropenium cation followed by a controlled hydrolysis ofthe resulting dichlorocyclopropene offers a convenient synthesis ofaromatic cyclopropenones (e.g., Poloukhtine et al., J. Org. Chem. 2003,68:7833-7840). Thus, the target cyclopropenone 5a was obtained bytreatment of 3,3′-bisbutoxybibenzyl (8) with tetrachlorocyclopropenonein the presence of aluminum chloride followed by in situ hydrolysis ofthe intermediate dichlorocyclopropene. In addition to 5a, a small amountof a bis-butoxy analog (5c) was isolated (FIG. 4) (see Examples).

To explore the utility of the “photo-click” chemistry for the temporaland spatial controlled labeling of live cells, we have prepared thebiotinylated cyclopropenone 5b (FIG. 4). Thus, cyclopropenone 5a wascoupled with diethylene glycol acetate under Mitsunobu conditions togive 5e in 92% yield. The carbonyl group of cyclopropenone 5e wasprotected as a neopentyl glycol acetal by treatment with neopentylglycol in the presence of BF₄O(C₂H₅)₃ and the acetyl ester of theresulting compound 10 was saponified with sodium methoxide in methanolto produce 11. Treatment of 11 with 4-nitrophenyl chlorofoimate gaveactivated intermediate 12 (FIG. 4), which was immediately reacted withN-Boc-N′-biotinyl-3,6-dioxaoctane-1,8-diamine to provide carbamate 13Finally, the acetal-protecting group of 13 was removed to give therequired cyclopropenone-biotin conjugate 5b by the treatment withAmberlyst 15 in acetone. The performance of the “photo-click” reagent 5bwas compared to a known labeling reagent 4b and to the independentlyprepared biotinylated dibenzocyclooctyne 6b (FIG. 5).

Dibenzocyclooctyne 6d was synthesized by the preparative photolysis ofcyclopropenone 5d. Conjugation of the former with a biotin groupfollowed procedures used in the conversion of acetal 10 into compound 13(FIG. 4).

The UV spectra of methanol solutions of cyclopropenones 5a-c contain twoclose-lying intense bands (λ_(max)=331 nm and 347 nm, log εapproximately 4.5, FIG. 6) (see Examples). Irradiation of 5a-c with 350nm light resulted in efficient (Φ₃₅₅=0.33) decarbonylation of thestarting material, which can be observed by bleaching of the 331-347 nmbands, and the quantitative formation of acetylenes 6a-c. Incubation ofsolutions of cyclopropenone 5a-c and benzyl- or phenyl azide in the darkfor several days did not result in detectable changes in UV absorbance.HPLC analysis of the mixture showed only the presence of startingmaterials. Upon irradiation of these solutions, however, the azidesrapidly reacted with photo-generated cycloalkyne 6a-c to produce thecorresponding triazoles 7a-c in quantitative yields. It should be notedthat the photoproducts 6a-c and 7a-c have virtually no absorbance above340 nm (FIG. 6; see Examples), thus allowing for selective irradiationof cyclopropenones 5a-c in their presence and for the convenientmonitoring of the reaction progress.

Kinetics of the cycloaddition reaction. The rate measurements ofcycloaddition of acetylenes 6c and 4a were conducted by UV spectroscopyat 25±0.1° C. A calculated amount of 0.25 M solutions of an aziderequired to achieve desired azide concentration (6×10⁻⁴-1.5×10⁻² M) wasadded to a thermally equilibrated ca. 6×10⁻⁵ M solution of acetylene inMeOH. Reactions were monitored by following the decay of thecharacteristic absorbance of acetylenes ca. 317 nm (FIG. 6). Consumptionof starting material followed a first order equation well and thepseudo-first order rate constants were obtained by the by least-squaresfitting of the data to a single exponential equation. The dependence ofthe observed rates on the concentration of azides was linear. Theleast-squares fitting of the data to a linear equation producedbimolecular rate constants summarized in Table 2. It was found that thismethod provides more accurate values of rate constants compared todetermination by NMR. Interestingly, the rate constants forcycloaddition of acetylene 6c with benzyl azide were very similar tothat of dibenzocyclooctynol (4a), and thus, the aromaticalkoxy-substitutents of 6a-c do not appear to influence the rateconstants.

TABLE 2 Bimolecular rate constants for the reaction of acetylene 4a and6c with azides in methanol Acetylene Azide Rate (M⁻¹ s⁻¹) 4a Benzylazide 5.67 × 10⁻² 6c Benzyl azide 7.63 × 10⁻² 6c n-Butyl azide 5.86 ×10⁻² 6c 1-Phenyl-2-azidopropane 3.43 × 10⁻² 6c Phenyl azide 1.63 × 10⁻²

Having established that light activation of cyclopropenones results inthe clean formation of the corresponding dibenzocyclooctynes, which canundergo cycloadditions with azides (e.g., metal-free cycloadditions inpreferred embodiments) to give corresponding triazoles, attention wasfocused on labeling living cells modified with azido groups. Thus,Jurkat cells were cultured in the presence of 25 mM of peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz) for 3 days to metabolicallyintroduce N-azidoacetyl-sialic acid (SiaNAz) groups into glycoproteinsand glycolipids. As a negative control, Jurkat cells were employed thatwere grown in the presence of peracetylated N-acetylmannosamine(Ac₄ManNAc). The cells were exposed to 30 micromolar of compound 4b, 5b,and 6b for 1 hour at room temperature. In addition, cells andcyclopropenone 5b were exposed to light (350 nm) for 1 minute to foamin-situ cyclooctyne 6b and then incubated for 1 hour at roomtemperature. Next, the cells were washed and stained withavidin-fluorescein isothiocyanate (FITC) for 15 minutes at 4° C. Theefficiency of the two-step cell surface labeling was determined bymeasuring the fluorescence intensity of the cell lysates. Cyclooctynes4b and 6b exhibited strong labeling of the cells (FIG. 7 a).Furthermore, in-situ activation of 5b to give 6b resulted in equallyefficient cell labeling. As expected, low fluorescence intensities weremeasured when cells were exposed to cyclopropenone 5b in the darkdemonstrating that this compound can be selectively activated by a shortirradiation with 350 nm light. Corresponding control cells showednegligible background labeling.

The concentration-dependency of the cell surface labeling was studied byincubating cells with various concentrations of 4b, in-situ activated5b, and 6b, followed by staining with avidin-FTIC (FIG. 7 b).

Jurkat cells were cultured in the presence of 25 mM of peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz) for 3 days to metabolicallyintroduce N-azidoacetylsialic acid (SiaNAz) groups into glycoproteins.As a negative control, Jurkat cells were employed that were grown in thepresence of peracetylated N-acetylmannosamine (Ac₄ManNAc). The cellswere exposed to 30 micromoles of compound 4b, 5b, and 6b for 1 hour at37° C. In addition, cells and cyclopropenone 5b were exposed to light(350 nm) for 1 minute to form in-situ cyclooctyne 6b and then incubatedfor 1 hour at 37° C. Next, the cells were washed and stained withavidin-fluorescein isothiocyanate (FITC) for 15 minutes at 4° C. and theefficiency of the two-step cell surface labeling was determined bymeasuring the fluorescence intensity of the cell lysates. Cyclooctynes4b and 6b exhibited strong labeling of the cells (FIG. 7 a).Furthermore, in-situ activation of 5b to give 6b resulted in equallyefficient cell labeling. As expected, low fluorescence intensities weremeasured when cells were exposed to cyclopropenone 5b in the darkdemonstrating that this compound can be selectively activated by a shortirradiation with 350 nm light. Corresponding control cells showednegligible background labeling. To ensure that light activation of 5bhad no effect on cell viability, cell morphology and exclusion of trypanblue were examined after exposure to UV light for 1 minute andfortunately no changes were observed compared to cells that were notexposed to UV light (data not shown).

The concentration-dependency of the cell surface labeling was studied byincubation cells with various concentrations of 4b, in-situ activated5b, and 6b, followed by staining with avidin-FTIC (FIG. 7 b). Asexpected, cells displaying azido groups showed a dose-dependent increasein fluorescence intensity. Reliable fluorescent labeling was achieved ata concentration of 3 micromolar, however, optimal results were obtainedat concentrations ranging from 10 to 100 micromolar. Interestingly, atlow concentration 6b gave a somewhat higher fluorescent reading than 4b.A time course experiment demonstrated that the labeling with 4b and 6bwas reaching completion at an incubation time of 60 minutes (FIG. 7 c).

To identify the nature of the azide-labeled Jurkat glycoconjugates, celllysates were analyzed by Western blot analysis (FIG. 8). Jurkat cellsgrown for 3 days in the presence of Ac₄ManNAc or Ac₄ManNAz wereincubated with compounds in-situ activated 5b or 6b (30 micromolar) andthen lysed. The Western blot was probed with an anti-biotin antibodyconjugated to horseradish peroxidase (HRP). Significant glycoproteinlabeling was only observed in lysates from cells grown in the presenceof Ac₄ManNAz. Furthermore, similar patterns of labeling were apparentafter incubation with in-situ activated 5b and 6b.

To ensure that in situ activation of 5b had no effect on cell viabilityand morphology, cells were assessed for the ability to exclude trypanblue and fortunately no changes were observed compared to cells thatwere not exposed to 5b both with and without UV light activation (FIG.9). Cell viability was also examined after incubation with 5b with andwithout light activation followed by reincubation for 5 hours (seeExamples). In both cases, the ability of the cells to reduce MTT to itsinsoluble formazan salt was negligible.

Next, attention was focused on visualizing azido-containingglycoconjugates of living cells by confocal microscopy. Thus, adherentChinese hamster ovary (CHO) cells were cultured in the presence ofAc₄ManNAz (100 μM) for three days. The resulting cell surface azidogroups were reacted with in situ generated 6b (30 μM) and thenvisualized with avidin-Alexa fluor 488. As expected, staining was onlyobserved at the cell surface (FIG. 10) and showed similar cell surfacelabeling as obtained by staining with 4b. Cells cultured in the presenceof Ac₄ManNAz (100 μM) exhibited very low fluorescence staining,confirming that background labeling is negligible. As expected, cellsmetabolically labeled with ManNAz and exposed to 5b in the dark showedalso negligible staining.

In conclusion, it has been shown that light activation of cyclopropenone5a-c results in the clean formation of the correspondingdibenzocyclooctyne 6a-c, which can undergo fast cycloadditions (e.g.,catalyst-free cycloadditions) with azides to give correspondingtriazoles. In-situ light activation of 5b made it possible toefficiently label living cells expressing glycoproteins containingN-azidoacetyl-sialic acid. The cyclopropenone-based “photo-click”chemistry reported here can provide greater bioorthogonality andversatility than recently developed reaction of alkenes with aphoto-generated nitrile imine (e.g., Song et al., J Am. Chem. Soc. 2008,130:9654-9655). It is to be expected that the properties of compoundssuch as 5b will make it possible to label living organisms in a temporaland spatial controlled manner (e.g., Pelliccioli et al., Photochem.Photobiol. Sci. 2002, 1:441-458; Mayer et al., Angew. Chem. Int. Ed.2006, 45:4900-4921; and Ellis-Davies, Nat. Methods 2007, 4:619-628).Furthermore, the hydroxy group in 5a can be easily esterified orconverted to an ether (e.g., 5d thus allowing for the attachment of the“photo-click” group to various substrates or surfaces. Compounds derivedfrom 5a can offer opportunities for temporal and spatial controlledligation (e.g., copper-free ligation in preferred embodiments), whichmay for example be attractive for microarray development. In addition tothis type of application, it is to be expected that other fields ofscience such the fabrication of microarrays and the preparation ofmultifunctional materials, may benefit from photo-click chemistry. Inthis respect, Cu-mediated click reactions have been used for thefabrication of saccharide microarrays by offering a convening approachto immobilize azide-modified saccharides to an alkyne-modified surface(e.g., Sun et al., Bioconjugate Chem. 2006, 17:52). It is to be expectedthat surface modification with compound 5a will offer an excitingopportunities for spatially controlled ligand immobilization using lightactivation followed by ligation (e.g., copper-free ligation in preferredembodiments). Furthermore, metal-free click reactions have been appliedin materials chemistry (e.g., Johnson et al., Chem. Commun. 2008,3064-3066; Lallana et al., J. Am. Chem. Soc. 2009, 131:5748; and Ingliset al., Angew. Chem. Int. Ed. Engl. 2009, 48:2411-2414), and the obviousadvantage of such a synthetic approach is that it offers a reliableapproach for macromolecule modification without the need of using toxicreagents. Therefore, it is to be expected that the combined use oftraditional- and photo-activated metal click reactions will offer anattractive approach for multi-functionalization of polymers andmacromolecules (e.g., Lundberg et al., Macromol. Rapid Comm. 2008,29:998-1015; Lutz, Angew. Chem. Int. Ed. Engl. 2007, 46:1018; andFournier et al., Chem. Soc. Rev. 2007, 36:1369-1380).

It should be emphasized that the embodiments of the present disclosure,particularly, any “preferred” embodiments, are merely possible examplesof the implementations, merely set forth for a clear understanding ofthe principles of the disclosure. Many variations and modifications maybe made to the above-described embodiment(s) of the disclosure withoutdeparting substantially from the spirit and principles of thedisclosure. All such modifications and variations are intended to beincluded herein within the scope of this disclosure, and the presentdisclosure and protected by the following claims.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere. The term “aqueous solution” as used herein refers to asolution that is predominantly water and retains the solutioncharacteristics of water. Where the aqueous solution contains solventsin addition to water, water is typically the predominant solvent.

EXAMPLES Example 1

Photophysical properties, generation, and reactivity ofdibenzocyclooctynes. Refer now to FIG. 11, and to FIG. 6. The UVspectrum, as shown in FIG. 6, of cyclopropenone 5c in methanol containedtwo close-lying bands (λ_(max)of about 331 nm and about 347 nm) ofsimilar intensity (log ε approximately 4.5; FIG. 6). Irradiation ofmethanol solutions of 5c with 350 nm (UV lamp) or 355 nm (Nd-YAG laser)light resulted in the efficient (Φ₃₅₅=0.33) decarbonylation ofcyclopropenone 5c, and the quantitative formation of the correspondingacetylene 6c, as shown in FIG. 11.

The photochemistry is very clean since no additional photoproducts weredetected in the photolysates. The acetylene 6c then rapidly reacted withalkyl- or aryl azide present in solution to give the triazole 7c. It isimportant to note that the absorbance bands of the acetylene 6c(λ_(max)=about 301 and about 317 nm, as shown in FIG. 6), and of thetriazole 7c (λ_(max)=about 310 nm, as shown in FIG. 6) are shifted tothe shorter wavelengths, in comparison to the starting material 5c. Boththe acetylene 6c and triazole 7c have virtually no absorbance aboveabout 340 nm. This feature allows for the selective irradiation ofcyclopropenone 5c and for the monitoring of the reaction progress.

Reaction of photochemically generated acetylene 6c with primary,secondary, benzyl, and aryl azides produces corresponding triazoles 7cin quantitative yields. No other products were detected by GC/MS or TLC.

Example 2

The rate measurements of the cycloaddition reaction of acetylene 6c withazides was conducted by UV spectroscopy following the decay of the 317nm band of 6c (as shown in FIG. 6) in the photolysate at variousconcentrations of azides from about 0.5 mM to about 20 mM). The reactionfollowed a first order equation, and the pseudo-first order rateconstants were obtained by the least-squares fitting of the data to asingle exponential equation. The dependence of the observed rates on theconcentration of azides was linear. The least-squares fitting of thedata to a linear equation produced bimolecular rate constants summarizedin Table 1.

TABLE 1 Bimolecular rate constants for the reaction of acetylene 6c withazides in methanol. Azide Rate (M⁻¹ s⁻¹) n-Butyl azide 5.86 × 10⁻²1-Phenyl-2-azidopropane 3.43 × 10⁻² Phenyl azide 1.63 × 10⁻² Benzylazide 7.63 × 10⁻²

The rate constants of the cycloaddition reaction of acetylene 6c withazides were found to be similar to the recently reported rate of thereaction of difluoro- (Baskin et al., (2007) Proc. Natl. Acad. Sci.U.S.A. 104: 16793-16797) and dibenzo-substituted (Boons et al., (2008)Angew. Chem., Int. Ed. 47: 2253-2255) cyclooctynes with benzyl azide(2-[(6,6-difluoro-4-cyclooctyn-1-yl)oxy]-acetic acid: 0.076 M⁻¹ s⁻¹; and11,12-didehydro-5,6-dihydro-dibenzo[a,e]cycloocten-5-ol: 0.0568 M⁻¹ s⁻¹.

Example 3

Substituents may be attached to the cyclopropenone precursor byreplacement of one of the butoxy groups with an appropriate linker Todemonstrate this strategy cyclopropenone 5a was prepared (as shown inFIG. 12). The hydroxy group of the latter may be readily converted intoan ether or an ester. Thus, the reaction of 5a with butanol ordiethylene glycol acetate in the presence of PPh₃ and DEAD at 0° C.produced 5c and 5d in a good yield (see FIG. 12).

The reaction of 5a with a carboxylic acid in the presence of DCC andcatalytic amount of DMAP provided ester 5e in 68% yield. Diethyleneglycol-derivatized cyclopropenone 5d was further linked to biotin,producing the biotin-cyclopropenone conjugate 5b as shown in FIG. 12.

Diphenyl cyclopropenones, such as 5a-5e, had long shelf lives, and couldwithstand elevated temperatures. Thus, the parent diphenylcyclopropenonewas quantitatively recovered after stirring for 5 hours in DMSO at 130°C. (Poloukhtine & Popik (2003) J. Org. Chem. 68: 7833-7840). Thecyclopropenones are also stable in solution in the absence of light. Forexample, cyclopropenone 5c showed no decomposition after incubation for3 days at 40° C. in aqueous and methanol solutions.

Significantly, the cyclopropenones 5a-5e do not react with azides atroom temperature.

Example 4

Synthesis of cyclopropenone 5c and 5a. A central step in the preparationof the cyclopropenones 5c and 5a is a double Friedel-Crafts reaction of3,3′-dibutoxybibenzyl 8 with tetrachlorocyclopropenone in the presenceof anhydrous aluminum trichloride under medium dilution conditions(about 0.05 M in methylene chloride, as shown in FIG. 13).

The mono-hydroxy substituted cyclopropenone 5a was the major product ofthis reaction. Formation of bis-butoxy derivative 5c depended on thereaction conditions. Thus, after overnight incubation of the reactionmixture at room temperature, only 5a was isolated. However, incubationfor only 5 hours resulted in formation of both 5c and 5a in 1:2 ratio.

Examples 5

General Procedures

All NMR spectra were recorded in CDCl₃ and referenced to TMS unlessotherwise noted. Melting points are uncorrected. Purification ofproducts by column chromatography was performed using 40-63 micrometersilica gel. Tetrahydrofuran was distilled from sodium/benzophenoneketyl; ether and hexanes were distilled from sodium. Other reagents wereobtained from Aldrich or VWR and used as received unless otherwisenoted.

Materials

11,12-didehydro-5,6-dihydro-dibenzo[a,e]cycloocten-5-ol (4a) and11,12-didehydro-5,6-dihydrodibenzo [a,e]cycloocten-5-yl ester of19-[(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]-15-oxo-5,8,11-trioxa-2,14-diazanonadecanoicacid (4b) were prepared as reported previously (Ning et al., Angew.Chem. Int. Ed. 2008, 47:2253-2255).

1,2-Bis(3-butoxyphenyl)ethane (8). BBr₃ (11.3 g, 45 mmol) was added to asolution of 1,2-bis(3-methoxyphenyl)ethane (Brunner et al., Inorg. Chim.Acta 2003, 350:39-48; 11.56 g; 47.8 mmol) in CH₂Cl₂ at −78° C. Thereaction mixture was slowly warmed to room temperature, and stirredovernight. The reaction mixture was quenched with water, diluted withCH₂Cl₂, and the reaction mixture extracted with 2 M solution of NaOH(3×100 mL). The aqueous layer was slowly acidified at 0° C. withconcentrated HCl to approximately pH=1, the grey precipitate wasfiltered, washed with water, dried in the air at room temperature, andthen under vacuum at 85° C. over 5 hours to provide 10.3 g of crude1,2-bis(3-hydroxyphenyl)ethane as grey solid.

A suspension of crude 1,2-bis(3-hydroxyphenyl)ethane (10.3 g), BuBr(6.50 g, 143.4 mmol), and K₂CO₃ (20.08 g, 143.4 mmol) in DMF (70 mL) wasstirred overnight at 75° C., cooled to room temperature, diluted withhexanes (approximately 150 mL) and water (approximately 250 mL) Theorganic layer was separated, washed with water, brine, dried overanhydrous MgSO₄, and concentrated. The residue was separated bychromatography (Hex:EtOAc 40:1) to provide 11.22 g, (72%, 34.42 mmol) of1,2-bis(3-butoxyphenyl)ethane as slightly yellow oil that slowlycrystallizes on standing. ¹H NMR: 7.18 (dt, J=8.8, 1.2 Hz, 2 H), 6.77(d, J=8.0 Hz, 2 H), 6.75-6.70 (m, 4 H), 3.93 (t, J=6.4 Hz, 4 H), 2.88(s, 4 H), 1.75 (5, J=6.4 Hz, 4 H), 1.48 (sex, J=7.2 Hz, 4 H), 0.98 (t,J=6.8 Hz, 6 H); ¹³C NMR: 159.4, 143.6, 129.5, 120.9, 115.0, 112.1, 67.8,38.1, 31.6, 19.5, 14.1; MS calc for C₂₂H₃₀O₂ (M⁺) 326.2246, EI-HRMSfound 326.2280.

4-Butoxy-9-hydroxy-6,7-dihydro-1H-dibenzo[a,e]cyclopropa-[c][8]annulen-1-one(5a), and4,9-dibutoxy-6,7-dihydro-1H-dibenzo[a,e]cyclopropa[c][8]annulen-1-one(5c). Tetrachloro-cyclopropene was added to a suspension of AICl₃ (2.45g, 13.76 mmol) in CH₂Cl₂ (200 mL), the reaction mixture was stirred for10 minutes at room temperature, and then cooled to −78° C. A solution of8 (4.48 g, 13.76 mmol) in CH₂Cl₂ (approximately 10 mL) was addeddropwise, and the reaction mixture was stirred for approximately 2 hoursat −78° C., slowly warmed to room temperature, and stirred for an extrahour at room temperature. The reaction was quenched by 5% aqueous HClsolution, the organic layer was separated, washed with water, dried overanhydrous MgSO₄, and concentrated. The residue was separated bychromatography (CH₂Cl₂:MeOH 20:1) to provide 0.997 g (3.12 mmol, 23%) of5a as yellow powder and 0.628 g (1.67 mmol, 12%) of 5c as white powder.

5a. ¹ H NMR (DMSO): 10.41 (s, 1 H), 7.73 (d, J=8.4 Hz, 1 H), 7.66 (d,J=8.4 Hz, 1 H), 7.05 (d, J=2.4 Hz, 1 H), 6.97 (dd, J=8.8, 2.4 Hz, 1 H),6.86 (d, J=2.4 Hz, 1 H), 6.80 (dd, J=8.4, 2.4 Hz, 1 H), 4.05 (t, J=6.4Hz, 2 H), 3.42-3.35 (m, 1 H) 2.45-2.35 (m, 3 H), 1.69 (p, J=7.2 Hz, 2H), 1.41 (sex, J=7.6 Hz, 2 H), 0.91 (t, J=7.2 Hz, 3 H); ¹³C NMR: 158.9,155.42, 155.19, 155.07, 127.1, 126.9, 117.5, 116.96, 116.72, 116.1,113.3, 112.1, 110.79, 110.34, 68.1, 36.8, 36.7, 31.5, 19.5, 14.1. MScalc for C₂₁H₂₁O₃ (MH⁺) 321.1491, APCI-HRMS found 321.1482.

5c. ¹H NMR: 7.73 (d, J=9.6 Hz, 2 H), 6.69 (m, 4 H), 4.04 (t, J=6.0 Hz, 4H), 3.33 (d, J=10.4 Hz, 2 H), 2.63 (d, J=10.4 Hz, 2 H), 1.80 (p, J=6.0Hz, 4 H), 1.52 (s, J=7.6 Hz, 4 H), 1.00 (t, J=7.6 Hz, 6 H); ¹³C NMR:162.3, 154.0, 148.0, 142.3, 136.0, 116.5, 112.5, 68.2, 37.4, 31.4,19.42, 14.03.

2-[2-(9-Butoxy-6,7-dihydro-1H-dibenzo[a,e]cyclopropa[c][8]annulen-1-one)ethoxy]ethylacetate (5d). A solution of DEAD (0.635 g, 3.75 mmol) in THF(approximately 5 mL) was added to a solution of 5a (0.75 g, 2.34 mmol),PPh₃ (0.983 g, 3.75 mmol), and 2-(2-hydroxyethoxy)ethyl acetate (0.44 g,3.0 mmol) in THF (100 mL) at room temperature, and the reaction mixturewas stirred for 30 minutes at room temperature. Solids were separated byfiltration, solvents were removed in vacuum, and the residue separatedby chromatography (Hex:EtOAc 2:1→Hex:EtOAc:CH₂Cl₂ 4:3:1→Hex:EtOAc:CH₂Cl₂5:5:4+5% of MeOH) to produce 0.971 g (2.16 mmol, 92%) of 5d as slightlyyellow oil that crystallizes on standing. ¹H NMR: 7.93 (d, J=8.4 Hz, 2H), 6.94-6.86 (m, 4 H), 4.27 (t, J=4.4 Hz, 2 H), 4.22 (t, J=4.4 Hz, 2H), 4.04 (t, J=6.0 Hz, 2 H), 3.90 (t, J=4.4 Hz, 2 H), 3.72 (t, J=4.4 Hz,2 H), 3.33 (d, J=10.4 Hz, 2 H), 2.62 (d, J=11.2 Hz, 2 H), 2.09 (s, 3 H),1.80 (p, J=7.2 Hz, 2 H), 1.52 (sex, J=7.6 Hz, 2 H), 1.00 (t, J=7.2 Hz, 3H); ¹³C NMR: 171.3, 162.1, 161.5, 153.5, 147.81, 147.78, 142.5, 135.8,135.7, 116.7, 116.4, 116.36, 116.13, 112.32, 112.30, 69.43, 69.39, 68.0,67.6, 63.5, 37.2, 31.1, 21.0, 19.2, 13.8.

2-{2-[(9-Butoxy-5′,5′-dimethyl-6,7-dihydrospiro-[dibenzo[a,e]cyclopropa[c][8]annulene-1,2′-[1,3]dioxan]-4-yl)oxy]ethoxy}ethylacetate (10). BF₄O(C₂H₅)₃ (0.45 g, 2.38 mmol, 1.1 eq) was added to asolution of cyclopropenone 5d (0.971 g, 2.16 mmol) in CH₂Cl₂ (5 mL) atroom temperature, and the resulting solution was stirred for 20 minutesat room temperature. A solution of neopentyl glycol (0.270 g, 2.59 mmol,1.2 eq) and Et₃N (0.330 g, 3.24 mmol, 1.5 eq) in CH₂Cl₂ (approximately1.5 mL) was added at room temperature, the reaction mixture was stirredfor 20 minutes at room temperature, and solvents were removed underreduced pressure. The residue was separated by chromatography (Hex:EtOAc5:1+1.5% of Et₃N →Hex:EtOAc 1:1+1.5% of Et₃N →Hex:EtOAc:CH₂Cl₂ 5:5:4+5%of MeOH and 1.5% of Et₃N) to provide 0.593 g (1.11 mmol, 96% calculatedon consumed substrate) of cyclopropenone acetal 10 as slightly yellowoil, and 0.431 g (0.96 mmol) of unreacted cyclopropenone 5d. ¹H NMR:7.65 (dd, J=8.4, 2.4 Hz, 2 H), 6.92-6.82 (m, 4 H), 4.26 (t, J=4.4 Hz, 2H), 4.18 (t, J=4.4 Hz, 2 H), 4.00 (t, J=6.4 Hz, 2 H), 3.9a (m, 4 H),3.88 (t, J=4.4 Hz, 2 H), 3.78 (t, J=4.4 Hz, 2 H), 3.24 (d, J=10.4 Hz, 2H), 2.41 (d, J=11.2 Hz, 2 H), 2.08 (s, 3 H), 1.79 (p, J=7.2 Hz, 2 H),1.51 (sex, J=7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19 (s, 3 H), 0.99 (t, J=7.2Hz, 3 H); ¹³C NMR: 171.1, 159.6, 159.0, 147.1, 131.5, 131.4, 124.2,123.4, 119.5, 118.9, 116.05, 115.94, 111.97, 111.92, 83.9, 79.2, 69.6,69.4, 63.5, 36.9, 31.3, 30.6, 22.62, 22.59, 21.0, 19.2, 13.9.

2-{2-[(9-Butoxy-5′,5′-dimethyl-6,7-dihydrospiro-[dibenzo[a,e]cyclopropa[c][8]annulene-1,2′-[1,3]dioxan]-4-yl)oxy]ethoxy}ethanol(11). A solution of NaOH (1.2 mL, 1.2 mmol, 1 M aqueous solution) wasadded to solution of acetate 10 (0.593 g, 1.11 mmol) in MeOH:THF (10:3mL) at room temperature, and the reaction mixture was stirred for 30minutes at room temperature. The reaction mixture was partiallyconcentrated under reduced pressure, diluted with EtOAc (approximately25 mL) and water (approximately 10 mL), the organic layer was separated,washed with brine, and dried over anhydrous MgSO₄. Solvents wereevaporated under reduced pressure, and the residue was separated bychromatography (Hex:EtOAc:CH₂Cl₂ 3:2:1+1.5% of Et₃N) to provide 0.493 g(0.89 mmol, 81%) of alcohol 11 as slightly yellow oil that crystallizeson standing. ¹H NMR: 7.65 (dd, J=8.4, 2.4 Hz, 2 H), 6.92-6.82 (m, 4 H),4.18 (t, J=4.4 Hz, 2 H), 4.04 (t, J=6.4 Hz, 2 H), 3.92 (m, 4 H), 3.88(t, J=4.4 Hz, 2 H), 3.77 (t, J=4.4 Hz, 2 H), 3.68 (t, J=4.4 Hz, 2 H),3.24 (d, J=10.8 Hz, 2 H), 2.41 (d, J=10.8 Hz, 2 H), 1.76 (p, J=7.2 Hz, 2H), 1.50 (sex, J=7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19 (s, 3 H), 0.99 (t,J=7.2 Hz, 3 H); ¹³C NMR: 159.8, 159.2, 147.4, 131.84, 131.75, 131.67,131.57, 124.4, 123.6, 119.8, 119.1, 116.3, 116.2, 112.2, 84.1, 79.4,72.8, 69.8, 68.0, 76.7, 62.0, 37.1, 31.5, 30.8, 22.9, 19.4, 14.2.

2-{2-[(9-Butoxy-5′,5′-dimethyl-6,7-dihydrospiro-[dibenzo[a,e]cyclopropa[c][8]annulene-1,2′-[1,3]dioxan]-4-yl)oxy]ethoxy}ethyl4-nitrophenyl carbonate (12). A solution of alcohol 11 (0.439 g, 0.89mmol) and pyridine (0.25 g, 3.21 mmol) in CH₂Cl₂ (approximately 5 mL)was added to a solution of 4-nitrophenyl chlorofoimate (0.30 g, 1.49mmol) in CH₂Cl₂ (25 mL) at room temperature, and the reaction mixturewas stirred for 20 minutes at room temperature. Solvent was evaporatedunder reduced pressure, and the residue was separated by chromatography(Hex:EtOAc 4:1+1.5% of Et₃N) to provide 0.317 g (0.48 mmol 80%) of 12 asand 0.113 g (0.23 mmol) of starting 11. ¹H NMR: 8.25 (d, J=8.8 Hz, 2 H)7.65 (dd, J=8.4, 2.0 Hz, 2 H), 7.35, (d, J=9.2, 2 H), 6.92-6.82 (m, 4H), 4.43 (t, J=4.4 Hz, 2 H), 4.19 (t, J=6.4 Hz, 2 H), 3.98 (t, J=4.4 Hz,2 H), 3.92 (m, 7 H), 3.22 (d, J=10.8 Hz, 2 H), 2.43 (d, J=10.8 Hz, 2 H),1.75 (p, J=7.2 Hz, 2 H), 1.51 (sex, J=7.6 Hz, 2 H), 1.21 (s, 3 H), 1.19(s, 3 H), 0.98 (t, J=7.2 Hz, 3 H); ¹³C NMR: 159.9, 159.2, 155.7, 152.7,150.0 147.4, 145.6, 131.77, 131.63, 125.5, 124.6, 123.4, 122.0, 119.8,119.1, 116.25, 116.19, 112.2, 112, 15, 84.1, 79.4, 70.0, 69.1, 68.4,70.0, 67.8, 37.1, 31.5, 30.8, 22.87, 22.79, 19.5, 14.1.

2-{2-[(9-Butoxy-1-oxo-6,7-dihydro-1H-dibenzo[a,e]cyclopropa-[c[8]annulen-4-yl)oxy]ethoxy}ethyl{2-[2-(2-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]amino}ethoxy)ethoxy]ethyl}carbamate(5b). A solution of cyclopropenone acetal 12 (0.21 g, 0.312 mmol) in DMF(approximately 2 mL) was added to a solution of Et₃N (0.18 g, 1.75 mmol)and N-Boc-N′-biotinyl-3,6-dioxaoctane-1,8-diamine (Ning et al., Angew.Chem. Int. Ed. 2008, 47:2253-2255) (0.13 g, 0.35 mmol) in DMF (35 mL) atroom temperature. The reaction mixture was stirred overnight at roomtemperature, most solvent was evaporated under reduced pressure, and theresidue was passed thought a short silica gel column (CH₂Cl₂:MeOH25:1+1.5% of Et₃N) to provide 0.275 g of crude product 13 that was usedin next step without any further purification.

A suspension of crude cyclopropenone acetal 13 (0.199 g) and Amberlyst15 (0.10 g) in Me₂CO (10 mL) was stirred for 60 minutes at roomtemperature. Solids were removed by filtration, solvent was evaporatedunder reduced pressure, and the residue was separated by chromatography(CH₂Cl₂:MeOH 10:1) to provide 17 mg of cyclopropenone 5b as an amorphoussolid. ¹H NMR: 7.65 (dd, J=8.4, 3.0 Hz, 2 H), 6.93-6.87 (m, 4 H), 6.66(s, b, 1 H), 6.25, (s, b, 1 H) 5.61 (m, b, 1 H) 5.39 (s, b, 1 H) 4.48(m, b, 1 H), 4.30-4.24 (m, 4 H), 4.21 (t, J=5.0 Hz, 2 H), 4.05 (t, J=7.5Hz, 2 H), 3.88 (t, J=5.5 Hz, 2 H), 3.78 (m, 2 H), 3.60 (s, 4 H), 3.44(q, J=6.5 Hz, 2 H), 3.40-3.30 (m, 4 H), 3.18-3.1 (m, 3 H), 2.27 (dd,J=16.0, 6.0 Hz, 1 H), 2.73 (d, J=16.0 Hz, 1 H), 2.62 (d, J=14.0 Hz, 2H), 2.20 (t, J=9.0 Hz, 2 H), 2.19-2.02 (m, 4 H), 1.81 (p, J=8.5 Hz, 2H), 1.74-1.60 (m, 4 H), 1.51 (sex, J=9.0 Hz, 2 H), 1.46-1.4 (m, 2 H),1.36 (t, J=9 Hz, 2 H), 1.00 (t, J=9.5 Hz, 3 H); ¹³C NMR: 173.4, 163.8,162.2, 161.5, 156.5, 153.8, 147.86, 147.83, 142.5, 141.9, 135.85,135.76, 116.71, 116.4, 116.28, 116.14, 112.39, 112.34, 70.13, 70.07,69.99, 69.88, 69.4, 68.0, 67.7, 63.9, 62.8, 60.2, 55.5, 45.8, 40.8,40.5, 39.1, 37.20, 37.15, 35.8, 31.1, 28.13, 28.07, 25.5, 19.2, 13.8,8.6; MS calc for C₄₁H₅₆N₄O₉S (M⁺—CO+Na) 803.3666, ESI-HRMS found808.3677.

Independent Preparation of Biotinylated Acetylene 6b

2-{2-[(9-Butoxy-5,6-didehydro-11,12-dihydrodibenzo[a,e][8]annulen-2-yl)oxy]ethoxy}ethanol(15). A solution of cyclopropenone 5d (0.54 g, 1.35 mmol) in MeOH:THF(1:1 v:v, 60 mL) was irradiated with 350 nm lamps for ca. 20 minutes.Solution was concentrated to 10 mL under reduced pressure, and 1 Maqueous NaOH solution (1.68 mL, 1.68 mmol) was added to the reactionmixture and stirred at room temperature for approximately 30 minutes.Ethyl acetate was added to the reaction mixture, the organic layer wasseparated, washed with water, brine, dried over anhydrous MgSO₄ andsolvent removed in vacuum. The residue was separated by chromatography(EtOAc:Hex 1:1.5) to provide 0.375 g (0.99 mmol, 73%) of alcohol 15 asan amorphous white solid. ¹H NMR: 7.20 (dd, J=8.4, 0.8 Hz, 2 H), 6.87(dd, J=11.2 Hz, 2.0, 2 H), 6.75 (td, J=8.0, 2.4 Hz, 2 H), 4.15 (t, J=4.4Hz, 2 H), 3.97 (t, J=6.0 Hz, 2 H), 3.87 (t, J=4.4 Hz, 2 H), 3.76 (s, b,2 H), 3.68 (d, J=4.4 Hz, 2 H), 3.17 (d, J=10.4 Hz, 2 H), 2.43 (d, J=10.4Hz, 2 H), 2.31 (1 H), 1.77 (p, J=7.2 Hz, 2 H), 1.50 (sex, J=7.2 Hz, 2H), 0.98 (t, J=7.2 Hz, 3 H); ¹³C NMR: 158.9, 158.3, 155.1, 126.99,126.84, 117.05, 116.93, 116.10, 112.08, 112.05, 110.91, 110.39, 72.8,69.8, 68.0, 67.7, 62.0, 36.94, 36.77, 31.5, 19.5, 14.1, 14.01. MS calcfor C₂₄H₂₈O₄ (M⁺) 380.1988, EI-HRMS found 380.1982.

2-{2-[(9-Butoxy-5,6-didehydro-11,12-dihydrodibenzo[a,e][8]annulen-2-yl)oxy]ethoxy}ethyl3-nitrophenyl carbonate (16). A solution of pyridine (0.20 g, 2.60 mmol)in CH₂Cl₂ (approximately 1 mL) was added to a solution of alcohol 15(0.24 g, 0.63 mmol) and 4-nitrophenyl cholroformate (0.20 g, 1.00 mmol)in CH₂Cl₂ (5 mL) at room temperature, and the reaction mixture wasstirred for 3 Hours. Solvent was evaporated under reduced pressure, andthe residue was separated by chromatography (Hex:EtOAc 4:1) to provide0.34 g (0.63 mmol 99%) of 16 as slightly yellow oil. ¹H NMR: 8.25 (d,J=8.8 Hz, 2 H), 7.36 (d, J=9.2 Hz, 2 H), 7.19 (d, J=8.8 Hz, 2 H), 6.89(dd, J=14.0, 2.4 Hz, 2 H), 6.79-6.75 (m, 2 H), 4.47 (t, J=4.4 Hz, 2 H),4.18 (t, J=4.4 Hz, 2 H), 3.97 (t, J=6.6 Hz, 2 H), 3.92-3.88 (m, 4 H),3.17 (d, J=10.8 Hz, 2 H), 2.42 (d, J=10.8 Hz, 2 H), 1.77 (p, J=7.2 Hz, 2H), 1.49 (sex, J=7.2 Hz, 2 H), 0.98 (t, J=7.2 Hz, 3 H); ¹³C NMR: 158.9,158.3, 155.7, 155.13, 155.08, 152.7, 145.6, 127.0, 126.9, 112.1, 121.9,117.0, 116.97, 116.94, 112.15, 112.11, 112.00, 111.0, 110.3, 70.1, 69.1,68.5, 68.0, 67.8, 36.9, 36.7, 31.5, 19.5, 14.2, 14.0.

2-{2-[(9-Butoxy-5,6-didehydro-11,12-dihydrodibenzo[a,e][8]annulen-2-yl)oxy]ethoxy}ethyl{2-[2-(2-{[-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]amino}ethoxy)ethoxy]ethyl}carbamate (6b). A solution of carbonate 16 (0.15 g, 0.28mmol) in DMF (approximately 2 mL) was added to a solution of Et₃N (0.5g, 4.95 mmol) and N-Boc-N′-biotinyl-3,6-dioxaoctane-1,8-diamine (Ning etal., Angew. Chem. Int. Ed. 2008, 47:2253-2255) (0.01 g, 0.28 mmol) inDMF (10 mL) at room temperature. The reaction mixture was stirredovernight at room temperature, most solvent was evaporated under reducedpressure, and the residue was separated by chromatography on(CH₂Cl₂:MeOH 30:1) to provide 0.164 g (0.21 mmol, 75%) of 6b. ¹H NMR δ7.19 (d, J=8.4 Hz, 2H), 6.88 (dd, J=9.5, 2.5 Hz, 2H), 6.76 (td, J=8.2,2.5, 2H), 6.74-6.65 (m, 1H), 6.54 (s, b, 1H), 5.74 (s, b, 1H), 5.60 (s,b 1H), 4.49-4.43 (m, 1H), 4.29-4.22 (m, 3H), 4.16-4.10 (m, 2H), 3.97 (t,J=6.5, 2H), 3.87-3.81 (m, 2H), 3.76 (m, 2H), 3.59-3.48 (m, 7H), 3.42 (m,2H), 3.37-3.12 (m, 2H), 3.21-3.09 (m, 4H), 2.86 (dd, J=12.6, 4.7 Hz,1H), 2.72 (d, J=12.7, 1H), 2.42 (d, J=10.9, 2H), 2.21 (t, J=7.4, 2H),1.81-1.56 (m, 5H), 1.48 (sex, J=7.4 Hz, 2H), 1.44-1.36 (m, 2H), 1.32 (t,J=7.4 Hz, 1H), 0.98 (t, J=7.4, 3H); ¹³C NMR δ 173.4, 164.1, 158.7,158.1, 156.5, 154.8, 126.66, 126.63, 116.80, 116.72, 116.59, 115.8,111.91, 111.83, 110.67, 110.14, 70.09, 70.04, 69.95, 69.90, 69.80,69.54, 67.78, 67.52, 63.88, 61.80, 60.2, 55.6, 45.6, 40.8, 40.5, 39.1,36.63, 36.61, 35.9, 31.3, 28.22, 28.08, 25.6, 19.2, 13.8, 8.5.

General Procedure for Preparative Photolyses of Cyclopropenones 5

3,9-Dibutoxy-5,6-didehydro-11,12-dihydrodibenzo[a,e][8]annulen-2-yl(3c). A solution of cyclopropenone 5c (0.20 g, 0.532 mmol) in MeOH (20mL, 2.72×10⁻²M) was irradiated (4×350 nm) for 20 minutes at roomtemperature. The solvent was evaporated under vacuum, and the residuewas separated by column chromatography (Hex:EtOAc 1:20) to provide 0.160g (0.459 mmol, 86%) of 6c as slightly yellow oil. NMR: 7.19 (d, J=8.4Hz, 2 H), 6.87 (d, J=2.4 Hz, 2 H), 6.75 (dd, J=8.4, 2.4 Hz, 2 H), 3.97(t, J=6.4 Hz, 4 H), 3.18 (d, J=11.2 Hz, 2 H), 2.44 (d, J=11.2 Hz, 2 H),1.77 (p, J=7.2 Hz, 4 H), 1.52 (sex, J=7.2 Hz, 4 H), 0.98 (t, J=7.2 Hz, 6H); ¹³C NMR: 158.9, 155.1, 126.9, 116.9, 116.2, 112.0, 110.6, 68.0,36.9, 31.5, 19.5, 14.1.

General Procedure for the Preparation of Triazoles 7

A solution of 6c (0.5 mmol) and appropriate organic azide (0.75 mmol) inMeOH was stirred overnight at room temperature. The solvent wasevaporated under reduced pressure, and the excess of aizde was removedby chromatography on silica gel.

1-Phenyl-6,11-dibutoxy-8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole(7c, R′=Ph). NMR: 7.53 (d, J=8.8 Hz, 1 H), 7.39 (s, 5 H), 6.85 (d, J=2.4Hz, 1 H), 6.79 (dd, J=8.4, 2.4 Hz, 1 H), 6.74 (d, J=2.4 Hz, 1 H), 6.62(d, J=8.8 Hz, 1 H), 6.51 (dd, J=8.4, 2.8 Hz, 1 H), 3.94 (t, J=6.4 Hz, 2H), 3.89 (t, J=6.4 Hz, 2 H), 3.50-3.30 (m, 2 H), 3.17-2.92 (m, 2 H),1.78-1.68 (m, 4 H), 1.46 (sep, J=7.2 Hz, 4 H), 0.96 (t, J=7.2 Hz, 3 H),0.95 (t, J=7.2 Hz, 3 H); ¹³C NMR: 159.9, 159.2, 147.0, 142.5, 139.7,137.0, 133.6, 133.0, 131.8, 129.5, 128.8, 124.8, 122.5, 118.8,116.5115.8, 112.8, 112.6, 67.81, 67.77, 36.2, 34.2, 31.5, 19.47, 19.45,14.10, 14.07.

6,11-Dibutoxy-1-butyl-8,9-dihydro-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazole(7c, R′=n-Bu) NMR: 7.43 (d, J=8.4 Hz, 1 H), 7.06 (d, J=8.4 Hz, 1 H),6.87 (d, J=2.4 Hz, 1 H), 6.78 (dd, J=8.4, 2.4 Hz, 1 H), 6.75 (dd, J=8.4,2.4 Hz, 1 H) 6.67 (d, J=2.4 Hz, 1 H), 4.42-4.24 (m, 2 H), 3.96 (t, J=6.4Hz, 2 H), 3.93 (t, J=6.8 Hz, 2 H), 3.40-3.32 (m, 1 H), 3.14-2.98 (m, 2H), 2.88-2.78 (m, 1 H), 1.86-1.68 (m, 6 H), 1.54-1.41 (m, 4 H),1.34-1.18 (m, 2 H), 0.98 (t, J=7.6 Hz, 3 H), 0.95 (t, J=7.2 Hz, 3 H),0.85 (t, J=7.2 Hz, 3 H); ¹³C NMR: 160.1, 158.9, 146.6, 143.3, 139.2,133.6, 133.2, 130.2, 122.8, 118.9, 116.6, 115.9, 112.9, 112.567.9, 67.7,48.2, 36.9, 33.4, 32.3, 31.55, 31.50, 19.8, 19.5, 14.1, 13.7.

Kinetic Experiments

Rate measurements were performed using Carry-300 Bio UV-Vis spectrometerequipped with a thermostattable cell holder. The temperature wascontrolled with 0.1° C. accuracy. A solution of 6c in MeOH (ca. 6×10⁻⁵M)in 1 cm quarts cell was thermally equilibrated for at least 30 minutesat 25±0.1° C. A calculated amount of 0.25 M solutions of an aziderequired to achieve desired azide concentration (6×10⁻⁴−1.5×10⁻²M) wasadded at once. Reactions were monitored by following the decay of thecharacteristic absorbance of acetylene 6c at approximately 317 nm. (FIG.6). The reaction follows a first order equation well and thepseudo-first order rate constants were obtained by the by least-squaresfitting of the data to a single exponential equation. The dependence ofthe observed rates on the concentration of azides was linear. Theleast-squares fitting of the data to a linear equation producedbimolecular rate constants summarized in Table 3.

TABLE 3 Bimolecular rate constants for the reaction of acetylene 6c withazides in methanol. Azide Rate (M⁻¹ s⁻¹) n-Butyl azide 5.86 × 10⁻²1-Phenyl-2-azidopropane 3.43 × 10⁻² Phenyl azide 1.63 × 10⁻² Benzylazide 7.63 × 10⁻²General Conditions for Biological Experiments

Synthetic compounds 4b, 5b, and 6b were reconstituted in DMF and storedat −80° C. Final concentrations of DMF never exceeded 0.56% to avoidtoxic effects. For the in situ photo-activation of biotinylatedcyclopropenone 5b mini-photoreactor available under the tradedesignation RAYONET equipped with 350 nm florescent tubes was employed.

Cell Culture Conditions

Human Jurkat cells (Clone E6-1; ATCC) were cultured in RPMI 1640 medium(ATCC) with L-glutamine (2 mM), adjusted to contain sodium bicarbonate(1.5 g/L), glucose (4.5 g/L), HEPES (10 mM), and sodium pyruvate (1 mM)and supplemented with penicillin (100 u/ml)/streptomycin (100micrograms/mL; Mediatech) and fetal bovine serum (FBS, 10%; Hyclone).Chinese hamster ovary (CHO) cells (Clone K1; ATCC) were cultured inKaighn's modification of Ham's F-12 medium (F-12K) with L-glutamine (2mM), adjusted to contain sodium bicarbonate (1.5 g L⁻¹) and supplementedwith penicillin (100 u mL⁻¹)/streptomycin (100 micrograms mL⁻¹) and FBS(10%). Cells were maintained in a humid 5% CO₂ atmosphere at 37° C.

Cell Surface Azide Labeling

Jurkat cells were seeded at a density of 75,000 cells mL⁻¹ in a totalvolume of 40 mL culture medium in the presence of peracetylatedN-azidoacetylmarmosamine (Ac₄ManNaz; 25 micromolar final concentration)and grown for 3 days, leading to the metabolic incorporation of thecorresponding N-azidoacetyl sialic acid (SiaNAz) into their cell surfaceglycoproteins. Control cells were grown in the presence of peracetylatedN-acetylmannosamine (Ac₄ManNac; 25 micromolar final concentration) for 3days. Similarly, CHO cells were grown for 3 days in the presence ofAc₄ManNaz (100 micromolar final concentration) or Ac₄ManNac (100micromolar final concentration).

Click Chemistry and Detection by Fluorescence Intensity

Jurkat cells bearing azides and control cells were washed with labelingbuffer (DPBS, pH 7.4 containing 1% FBS and 1% BSA), transferred to roundbottom tubes (1×10⁶ cells/sample) and incubated with the biotinylatedcompounds 4b, 5b, or 6b (0-100 micromolar) in labeling buffer for 0-90minutes at room temperature. To activate 5b in situ, immediately afteradding the compound to the cells, the cell suspension was subjected toUV light (350 nm) for 1 minute. The cells were washed three times withcold labeling buffer and then incubated with avidin conjugated withfluorescein (0.5 microgram/ml; Molecular Probes) for 15 minutes at 4° C.Following three washes and cell lysis in passive lysis buffer availablefrom PROMEGA (Promega), cell lysates were analysed for fluorescenceintensity (485 ex/520 em) using a microplate reader (BMG Labtech). Datapoints were collected in triplicate and are representative of threeseparate experiments. Fluorescence of Jurkat cell lysates was expressedas fluorescence (arbitrary units; AU) per 800,000 cells.

Measurement of Cytotoxicity

Cell viability and cell morphology were assessed by exclusion of trypanblue followed by microscopic evaluation immediately afterphotoactivation or after reincubation of the labeled cells in cellculture medium for 5 hours or overnight. Viability was measured byquantifying the cellular ability to reduce the water-soluble tetrazoliumdye 3-4,5-dimethylthiazole-2,5-diphenyl tetrazolium bromide (MTT) to itsinsoluble formazan salt (e.g., Sgouras et al., J. Mater. Sci.: Materialsin Medicine 1990, 1:61-68). Data points were collected in triplicate andexpressed as normalized values for control cells (100%).

Western Blot Analysis

Jurkat cells were harvested by centrifugation (5 minutes at 1,400 rpm)and resuspended as 5×10⁶ cells/mL The cell suspensions (250 microlitersper sample) were incubated with biotin-conjugated alkynes 4b and 6b andbiotin-conjugated cyclopropenone 5b (30 micromolar) or without compoundas control for 1 hour. To activate 5b in situ, immediately after addingthe compound to the cells, the cell suspension was subjected to UV light(350 nm) for 1 minute. The cells were washed (4×10 minutes) with coldDPBS, pH 7.4 containing FBS (1%) and lysed in passive lysis buffer. Thecell lysates were clarified by centrifugation at 15,000 rpm for 15minutes and the total protein content of the clear supernatants wasassessed using the bicinchonic acid assay (BCA; Pierce Biotechnology).Cell lysate samples (20 micrograms protein) in SDS-PAGE sample buffercontaining 2-mercaptoethanol were boiled for 5 minutes, resolved on a4-20 % Tris-HCl gel available from BIO-RAD and transferred tonitrocellulose membrane. Next the membrane was blocked in blockingbuffer (non-fat dry milk (5%; available from BIO-RAD) in PBST (PBScontaining 0.1% Tween-20 and 0.1% Triton X-100)) for 2 hours at roomtemperature. The blocked membrane was incubated for 1 hour at roomtemperature with an anti-biotin antibody conjugated to horseradishperoxidase (HRP) (1:100,000; Jackson ImmunoResearch Lab, Inc.) inblocking buffer and washed with PBST (4×10 minutes). Final detection ofHRP activity was performed using ECL Plus chemiluminescent substrateavailable under the trade designation Amersham), exposure to filmavailable from KODAK and development using a digital X-ray imagingmachine available from KODAK. The gel was stained by Coomassie toconfirm total protein loading.

Detection of Cell Labeling by Fluorescence Microscopy.

CHO cells bearing azides and untreated control cells were transferred toglass coverslips and cultured for 36 hours in their original medium.Live CHO cells were treated with the biotinylated compound 5b (30micromolar) in labeling buffer (DPBS, supplemented with FBS (1%)) for 1hour at room temperature. To activate 5b in situ, immediately afteradding the compound to the cells, the cells were subjected to UV light(350 nm) for 1 minute. Next, the cells were incubated with avidinconjugated with Alexa Fluor 488 (Molecular Probes) for 15 minutes at 4°C. Cells were washed 3 times with labeling buffer and fixed withformaldehyde (3.7% in PBS). The nucleus was labeled with the farred-fluorescent TO-PRO-3 dye (Molecular Probes). The cells were mountedwith an aqueous mounting medium available under the trade designationPERMAFLUOR (Thermo Electron Corporation) before imaging. Initialanalysis was performed on a Zeiss Axioplan2 fluorescent microscope.Confocal images were acquired using a 60× (NA1.42) oil objective. Stacksof optical sections were collected in the z dimensions. The step size,based on the calculated optimum for each objective, was between 0.25 and0.5 micrometers. Subsequently, each stack was collapsed into a singleimage (z-projection). Analysis was performed offline using ImageJ 1.39fsoftware (National Institutes of Health, USA) and ADOBE PHOTOSHOP CS3Extended Version 10.0 software (ADOBE SYSTEMS Incorporated), whereby allimages were treated equally.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

What is claimed is:
 1. A compound of the formula:

wherein: each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents O, NR⁶, or⁺N(R⁶)₂; each R⁴ is independently selected from the group consisting ofhydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, a C1-C10organic group, and a linking group; and each R⁶ is independentlyhydrogen, a C1-C10 organic group, or a linking group.
 2. The compound ofclaim 1 wherein each Ar is a phenyl group.
 3. The compound of claim 2wherein one or more carbon atoms of one or both phenyl groups aresubstituted with a substituent selected from the group consisting ofalkyl, aryl, heteroaryl, halogen, nitro, cyano, hydroxyl, alkoxyl,aryloxyl, thio, mercapto, alkylthio, arylthio, amino, alkylamino,arylamino, dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl,diarylaminocarbonyl, arylalkylaminocarbonyl, carboxyl, alkyloxycarbonyl,aryloxycarbonyl, aldehyde, arylcarbonyl, alkylcarbonyl, iminyl,aryliminyl, alkyliminyl, sulfo, alkylsulfonyl, arylsulfonyl,hydroximinyl, aryloximinyl, and alkoximinyl; and wherein two or morealkyl or heteroalkyl substituents of a phenyl group may optionally becombined to form fused aryl-alkyl or aryl-heteroalkyl ring systems. 4.The compound of claim 2 wherein one or more carbon atoms of one or bothphenyl groups are substituted with a PEGylated or biotinylated group. 5.The compound of claim 4 wherein the biotinylated group has the formula:


6. A compound of the formula:

wherein: each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents an oxygenatom; and each R⁴ is independently selected from the group consisting ofhydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, a C1-C10organic group, and a linking group.
 7. The compound of claim 6 whereineach Ar is a phenyl group.
 8. The compound of claim 7 wherein one ormore carbon atoms of one or both phenyl groups are substituted with asubstituent selected from the group consisting of alkyl, aryl,heteroaryl, halogen, nitro, cyano, hydroxyl, alkoxyl, aryloxy, thio,mercapto, alkylthio, arylthio, amino, alkylamino, arylamino,dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl,diarylaminocarbonyl, arylalkylaminocarbonyl, carboxyl, alkyloxycarbonyl,aryloxycarbonyl, aldehyde, arylcarbonyl, alkylcarbonyl, iminyl,aryliminyl, alkyliminyl, sulfo, alkylsulfonyl, arylsulfonyl,hydroximinyl, aryloximinyl, and alkoximinyl; and wherein two or morealkyl or heteroalkyl substituents of a phenyl group may optionally becombined to form fused aryl-alkyl or aryl-heteroalkyl ring systems. 9.The compound of claim 7 wherein one or more carbon atoms of one or bothphenyl groups are substituted with a PEGylated or biotinylated group.10. The compound of claim 9 wherein the biotinylated group has theformula:


11. A compound of the formula:

wherein: each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents an oxygenatom; R³ represents a organic group; and each R⁴ is independentlyselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, nitrate, nitrite, sulfate, a C1-C10 organic group, and a linkinggroup.
 12. The compound of claim 11 wherein each Ar is a phenyl group.13. The compound of claim 12 wherein one or more carbon atoms of one orboth phenyl groups are substituted with a substituent selected from thegroup consisting of alkyl, aryl, heteroaryl, halogen, nitro, cyano,hydroxyl, alkoxyl, aryloxyl, thio, mercapto, alkylthio, arylthio, amino,alkylamino, arylamino, dialkylamino, diarylamino, arylalkylamino,aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl, arylalkylaminocarbonyl,carboxyl, alkyloxycarbonyl, aryloxycarbonyl, aldehyde, arylcarbonyl,alkylcarbonyl, iminyl, aryliminyl, alkyliminyl, sulfo, alkylsulfonyl,arylsulfonyl, hydroximinyl, aryloximinyl, and alkoximinyl; and whereintwo or more alkyl or heteroalkyl substituents of a phenyl group mayoptionally be combined to form fused aryl-alkyl or aryl-heteroalkyl ringsystems.
 14. The compound of claim 12 wherein one or more carbon atomsof one or both phenyl groups are substituted with a PEGylated orbiotinylated group.
 15. The compound of claim 14 wherein thebiotinylated group has the formula:


16. A method of photochemically inducing the reaction of two materials,the method comprising: photochemically generating a cyclic alkyne from acyclopropenone of the formnula:

wherein each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents O, NR⁶, or⁺N(R⁶)₂; each R⁴ is independently selected from the group consisting ofhydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, a C1-C10organic group, and a linking group; and each R⁶ is independentlyhydrogen, a C1-C10 organic group, or a linking group; and contacting thecyclic alkyne with a material comprising an alkyne-reactive group underconditions effective for the cyclic alkyne and the material comprisingthe alkyne-reactive group to react.
 17. The method of claim 16 whereineach Ar is a phenyl group.
 18. The method of claim 17 wherein one ormore carbon atoms of one or both phenyl groups are substituted with asubstituent selected from the group consisting of alkyl, aryl,heteroaryl, halogen, nitro, cyano, hydroxyl, alkoxyl, aryloxyl, thio,mercapto, alkylthio, arylthio, amino, alkylamino, arylamino,dialkylamino, diarylamino, arylalkylamino, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl,diarylaminocarbonyl, arylalkylaminocarbonyl, carboxyl, alkyloxycarbonyl,aryloxycarbonyl, aldehyde, arylcarbonyl, alkylcarbonyl, iminyl,aryliminyl, alkyliminyl, sulfo, alkylsulfonyl, arylsulfonyl,hydroximinyl, aryloximinyl, and alkoximinyl; and wherein two or morealkyl or heteroalkyl substituents of a phenyl group may optionally becombined to form fused aryl-alkyl or aryl-heteroalkyl ring systems. 19.The method of claim 16 wherein the method photochemically induces theligation of the cyclic alkyne and the material comprising thealkyne-reactive group.
 20. The method of claim 16 wherein the methodforms a cyclic adduct.
 21. The method of claim 20 wherein the materialcomprising the alkyne-reactive group is a 1,3-dipole-functionalcompound.
 22. The method of claim 21 wherein the cyclic adduct is aheterocyclic compound.
 23. The method of claim 22 wherein the1,3-dipole-functional compound is selected from the group consisting ofazide-functional compounds, nitrile oxide-functional compounds,nitrone-functional compounds, azoxy-functional compounds, acyldiazo-functional compounds, and combinations thereof.
 24. The method ofclaim 23 wherein the 1,3-dipole-functional compound is anazide-functional compound, and the cyclic adduct is a triazole.
 25. Themethod of claim 24 wherein the azide-functional compound or the cyclicalkyne is bound to the surface of a substrate or integrated into asubstrate layer.
 26. The method of claim 25 wherein the substrate is asolid substrate or a cell membrane.
 27. The method of claim 25 whereinthe azide-functional compound is bound to the surface of a substrate orintegrated into a substrate layer, and the cyclic alkyne is a ligandthat binds to the azide.
 28. The method of claim 27 wherein the azideligand or the cyclic alkyne ligand is a detectable label.
 29. The methodof claim 24 wherein the azide-functional compound is selected from thegroup consisting of: an alkyl azide, a heteroalkyl azide, a cycloalkylazide, a heterocycloalkyl azide, an alkylamino azide, a benzyl azide, anaryl azide, an alkyacyl azide, and an arylacyl azide.
 30. The method ofclaim 24 wherein photochemically generating the cyclic alkyne comprisesirradiating the cyclopropenone with light having a wavelengthselectively absorbed by the cyclopropenone, and substantially notabsorbed by the cyclic alkyne or by the trizaole.
 31. The method ofclaim 30 wherein the wavelength of light is from about 220 nm to about450 nm.
 32. The method of claim 24 wherein the triazole has the foimula:

wherein: each Ar is a group independently representing a monocyclic orpolycyclic, aromatic or heteroaromatic ring; E represents O, NR⁶, or⁺N(R⁶)₂; R³ represents an organic group; each R⁴ is independentlyselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, nitrate, nitrite, sulfate, a C1-C10 organic group, and a linkinggroup; and each R⁶ is independently hydrogen, a C1-C10 organic group, ora linking group.
 33. The method of claim 20 wherein the materialcomprising the alkyne-reactive group is a diene, and the cyclic adductis a Diels-Alder adduct.
 34. The method of claim 20 wherein the materialcomprising the alkyne-reactive group is a nitrosoarene, and the cyclicadduct is an N-hydroxy indole.
 35. The method of claim 20 wherein thematerial comprising the alkyne-reactive group is a metal-containingcompound.
 36. The method of claim 35 wherein the metal-containingcompound is a four- or five-memebered platinacycle.
 37. The method ofclaim 20 wherein the material comprising the alkyne-reactive group iscarbon monoxide, the method further comprising contacting the cyclicalkyne with an alkene, and the cyclic adduct is a [2+2+1] cycloadditionproduct.
 38. The method of claim 16 wherein the material comprising thealkyne-reactive group is a metal carbene complex, the method furthercomprising contacting the cyclic alkyne with an alkene under conditionseffective to form a butadiene.
 39. The method of claim 36 wherein themethod comprises enyne metathesis.
 40. The method of claim 16 whereinthe material comprising the alkyne-reactive group is an alkyne, themethod further comprising contacting the cyclic alkyne with a metalcatalyst under conditions effective to form a different alkyne.
 41. Themethod of claim 40 wherein the method comprises alkyne metathesis. 42.The method of claim 17 wherein one or more carbon atoms of one or bothphenyl groups are substituted with a PEGylated or biotinylated group.43. The method of claim 42 wherein the biotinylated group has theformula:


44. The method of claim 25 wherein the cyclic alkyne is bound to thesurface of a substrate or integrated into a substrate layer, and theazide-functional compound is a ligand that binds to the cyclic alkyne.